Modulators of cystic fibrosis transmembrane conductance regulator

ABSTRACT

The present invention relates to modulators of cystic fibrosis transmembrane conductance regulator (“CFTR”), compositions thereof, and methods therewith. The present invention also relates to methods of treating diseases using modulators of CFTR.

CLAIM OF PRIORITY

The present application is a division of U.S. Ser. No. 12/605,053, filedOct. 23, 2009, which claims the benefit of priority under 35 U.S.C. §119to U.S. Provisional Application Ser. No. 61/107,830, filed Oct. 23, 2008and entitled “MODULATORS OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCEREGULATOR,” the entire contents of each of the above applications beingincorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to modulators of cystic fibrosistransmembrane conductance regulator (“CFTR”), compositions thereof, andmethods therewith. The present invention also relates to methods oftreating diseases using modulators of CFTR.

BACKGROUND OF THE INVENTION

ATP cassette transporters are a family of membrane transporter proteinsthat regulate the transport of a wide variety of pharmacological agents,potentially toxic drugs, and xenobiotics, as well as anions. They arehomologous membrane proteins that bind and use cellular adenosinetriphosphate (ATP) for their specific activities. Some of thesetransporters were discovered as multidrug resistance proteins (like theMDR1-P glycoprotein, or the multidrug resistance protein, MRP1),defending malignant cancer cells against chemotherapeutic agents. Todate, 48 such transporters have been identified and grouped into 7families based on their sequence identity and function.

One member of the ATP cassette transporters family commonly associatedwith disease is the cAMP/ATP-mediated anion channel, CFTR. CFTR isexpressed in a variety of cells types, including absorptive andsecretory epithelia cells, where it regulates anion flux across themembrane, as well as the activity of other ion channels and proteins. Inepithelial cells, normal functioning of CFTR is critical for themaintenance of electrolyte transport throughout the body, includingrespiratory and digestive tissue. CFTR is composed of approximately 1480amino acids that encode a protein made up of a tandem repeat oftransmembrane domains, each containing six transmembrane helices and anucleotide binding domain. The two transmembrane domains are linked by alarge, polar, regulatory (R)-domain with multiple phosphorylation sitesthat regulate channel activity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia lead to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhanced mucus accumulationin the lung and the accompanying microbial infections that ultimatelycause death in CF patients. In addition to respiratory disease, CFpatients typically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, more than 1000 diseasecausing mutations in the CF gene have been identified(http://www.genet.sickkids.on.ca/cftr/). The most prevalent mutation isa deletion of phenylalanine at position 508 of the CFTR amino acidsequence, and is commonly referred to as ΔF508-CFTR. This mutationoccurs in approximately 70 percent of the cases of cystic fibrosis andis associated with a severe disease.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia, leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dolmans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, R117H-CFTR and G551D-CFTR, other disease causing mutationsin CFTR that result in defective trafficking, synthesis, and/or channelgating, could be up- or down-regulated to alter anion secretion andmodify disease progression and/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions, chloride andbicarbonate) represents one element in an important mechanism oftransporting ions and water across the epithelium. The other elementsinclude the epithelial Na⁺ channel, ENaC, Na⁺/2Cl⁻/K⁺ co-transporter,Na⁺—K⁺-ATPase pump and the basolateral membrane K⁺ channels, that areresponsible for the uptake of chloride into the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ ion channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

Defective bicarbonate transport due to mutations in CFTR is hypothesizedto cause defects in certain secretory functions. See, e.g., “Cysticfibrosis: impaired bicarbonate secretion and mucoviscidosis,” Paul M.Quinton, Lancet 2008; 372: 415-417.

Mutations in CFTR that are associated with moderate CFTR dysfunction arealso evident in patients with conditions that share certain diseasemanifestations with CF but do not meet the diagnostic criteria for CF.These include congenital bilateral absence of the vas deferens,idiopathic chronic pancreatitis, chronic bronchitis, and chronicrhinosinusitis. Other diseases in which mutant CFTR is believed to be arisk factor along with modifier genes or environmental factors includeprimary sclerosing cholangitis, allergic bronchopulmonary aspergillosis,and asthma.

Cigarette smoke, hypoxia, and environmental factors that induce hypoxicsignaling have also been demonstrated to impair CFTR function and maycontribute to certain forms of respiratory disease, such as chronicbronchitis. Diseases that may be due to defective CFTR function but donot meet the diagnostic criteria for CF are characterized asCFTR-related diseases.

In addition to cystic fibrosis, modulation of CFTR activity may bebeneficial for other diseases not directly caused by mutations in CFTR,such as secretory diseases and other protein folding diseases mediatedby CFTR. CFTR regulates chloride and bicarbonate flux across theepithelia of many cells to control fluid movement, proteinsolubilization, mucus viscosity, and enzyme activity. Defects in CFTRcan cause blockage of the airway or ducts in many organs, including theliver and pancreas. Potentiators are compounds that enhance the gatingactivity of CFTR present in the cell membrane. Any disease whichinvolves thickening of the mucus, impaired fluid regulation, impairedmucus clearance, or blocked ducts leading to inflammation and tissuedestruction could be a candidate for potentiators.

These include, but are not limited to, chronic obstructive pulmonarydisease (COPD), asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, dry eye disease, and Sjögren's Syndrome,gastro-esophageal reflux disease, gallstones, rectal prolapse, andinflammatory bowel disease. COPD is characterized by airflow limitationthat is progressive and not fully reversible. The airflow limitation isdue to mucus hypersecretion, emphysema, and bronchiolitis. Activators ofmutant or wild-type CFTR offer a potential treatment of mucushypersecretion and impaired mucociliary clearance that is common inCOPD. Specifically, increasing anion secretion across CFTR mayfacilitate fluid transport into the airway surface liquid to hydrate themucus and optimized periciliary fluid viscosity. This would lead toenhanced mucociliary clearance and a reduction in the symptomsassociated with COPD. In addition, by preventing ongoing infection andinflammation due to improved airway clearance, CFTR modulators mayprevent or slow the parenchimal destruction of the airway thatcharacterizes emphysema and reduce or reverse the increase in mucussecreting cell number and size that underlyses mucus hypersecretion inairway diseases. Dry eye disease is characterized by a decrease in tearaqueous production and abnormal tear film lipid, protein and mucinprofiles. There are many causes of dry eye, some of which include age,Lasik eye surgery, arthritis, medications, chemical/thermal burns,allergies, and diseases, such as cystic fibrosis and Sjögrens'ssyndrome. Increasing anion secretion via CFTR would enhance fluidtransport from the corneal endothelial cells and secretory glandssurrounding the eye to increase corneal hydration. This would help toalleviate the symptoms associated with dry eye disease. Sjögrens'ssyndrome is an autoimmune disease in which the immune system attacksmoisture-producing glands throughout the body, including the eye, mouth,skin, respiratory tissue, liver, vagina, and gut. Symptoms, include, dryeye, mouth, and vagina, as well as lung disease. The disease is alsoassociated with rheumatoid arthritis, systemic lupus, systemicsclerosis, and polymypositis/dermatomyositis. Defective proteintrafficking is believed to cause the disease, for which treatmentoptions are limited. Modulators of CFTR activity may hydrate the variousorgans afflicted by the disease and may help to alleviate the associatedsymptoms. Individuals with cystic fibrosis have recurrent episodes ofintestinal obstruction and higher incidences of rectal polapse,gallstones, gastro-esophageal reflux disease, GI malignancies, andinflammatory bowel disease, indicating that CFTR function may play animportant role in preventing such diseases.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective ER processing of CFTR by the ERmachinery, has been shown to be the underlying basis not only for CFdisease, but for a wide range of other isolated and inherited diseases.The two ways that the ER machinery can malfunction is either by loss ofcoupling to ER export of the proteins leading to degradation, or by theER accumulation of these defective/misfolded proteins [Aridor M, et al.,Nature Med., 5(7), pp 745-751 (1999); Shastry, B. S., et al., Neurochem.International, 43, pp 1-7 (2003); Rutishauser, J., et al., Swiss MedWkly, 132, pp 211-222 (2002); Morello, J P et al., TIPS, 21, pp. 466-469(2000); Bross P., et al., Human Mut., 14, pp. 186-198 (1999)]. Thediseases associated with the first class of ER malfunction are cysticfibrosis (due to misfolded ΔF508-CFTR as discussed above), hereditaryemphysema (due to a1-antitrypsin; non Piz variants), hereditaryhemochromatosis, coagulation-fibrinolysis deficiencies, such as proteinC deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, Mucopolysaccharidoses (due to lysosomalprocessing enzymes), Sandhof/Tay-Sachs (due to β-hexosaminidase),Crigler-Najjar type II (due to UDP-glucuronyl-sialyc-transferase),polyendocrinopathy/hyperinsulemia, Diabetes mellitus (due to insulinreceptor), Laron dwarfism (due to growth hormone receptor),myleoperoxidase deficiency, primary hypoparathyroidism (due topreproparathyroid hormone), melanoma (due to tyrosinase). The diseasesassociated with the latter class of ER malfunction are Glycanosis CDGtype 1, hereditary emphysema (due to a1-Antitrypsin (PiZ variant),congenital hyperthyroidism, osteogenesis imperfecta (due to Type I, II,IV procollagen), hereditary hypofibrinogenemia (due to fibrinogen), ACTdeficiency (due to a1-antichymotrypsin), Diabetes insipidus (DI),neurophyseal DI (due to vasopvessin hormone/V2-receptor), neprogenic DI(due to aquaporin II), Charcot-Marie Tooth syndrome (due to peripheralmyelin protein 22), Perlizaeus-Merzbacher disease, neurodegenerativediseases such as Alzheimer's disease (due to βAPP and presenilins),Parkinson's disease, amyotrophic lateral sclerosis, progressivesupranuclear palsy, Pick's disease, several polyglutamine neurologicaldisorders such as Huntington's, spinocerebullar ataxia type I, spinaland bulbar muscular atrophy, dentatorubal pallidoluysian, and myotonicdystrophy, as well as spongiform encephalopathies, such as hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease (due to lysosomal α-galactosidase A), Straussler-Scheinkersyndrome (due to Prp processing defect), infertility pancreatitis,pancreatic insufficiency, osteoporosis, osteopenia, Gorham's Syndrome,chloride channelopathies, myotonia congenita (Thomson and Becker forms),Bartter's syndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, PrimaryCiliary Dyskinesia (PCD), PCD with situs inversus (also known asKartagener syndrome), PCD without situs inversus and ciliary aplasia,and liver disease.

Other diseases implicated by a mutation in CFTR include male infertilitycaused by congenital bilateral absence of the vas deferens (CBAVD), mildpulmonary disease, idiopathic pancreatitis, and allergicbronchopulmonary aspergillosis (ABPA). See, “CFTR-opathies: diseasephenotypes associated with cystic fibrosis transmembrane regulator genemutations,” Peader G. Noone and Michael R. Knowles, Respir. Res. 2001,2: 328-332 (incorporated herein by reference).

In addition to up-regulation of CFTR activity, reducing anion secretionby CFTR modulators may be beneficial for the treatment of secretorydiarrheas, in which epithelial water transport is dramatically increasedas a result of secretagogue activated chloride transport. The mechanisminvolves elevation of cAMP and stimulation of CFTR.

Although there are numerous causes of diarrhea, the major consequencesof diarrheal diseases, resulting from excessive chloride transport arecommon to all, and include dehydration, acidosis, impaired growth anddeath. Acute and chronic diarrheas represent a major medical problem inmany areas of the world. Diarrhea is both a significant factor inmalnutrition and the leading cause of death (5,000,000 deaths/year) inchildren less than five years old.

Secretory diarrheas are also a dangerous condition in patients withacquired immunodeficiency syndrome (AIDS) and chronic inflammatory boweldisease (IBD). Sixteen million travelers to developing countries fromindustrialized nations every year develop diarrhea, with the severityand number of cases of diarrhea varying depending on the country andarea of travel.

Accordingly, there is a need for potent and selective CFTR potentiatorsof wild-type and mutant forms of human CFTR. These mutant CFTR formsinclude, but are not limited to, ΔF508del, G551D, R117H, 2789+5G→A.

There is also a need for modulators of CFTR activity, and compositionsthereof, which can be used to modulate the activity of the CFTR in thecell membrane of a mammal.

There is a need for methods of treating diseases caused by mutation inCFTR using such modulators of CFTR activity.

There is a need for methods of modulating CFTR activity in an ex vivocell membrane of a mammal.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are useful asmodulators of CFTR activity. The compounds have the general Formula (I):

or pharmaceutically acceptable salts thereof, wherein R¹, R², R³ and Aare described generally and in classes and subclasses below.

These compounds and pharmaceutically acceptable compositions are usefulfor treating or lessening the severity of a variety of diseases,disorders, or conditions associated with mutations in CFTR.

DETAILED DESCRIPTION OF THE INVENTION General Description of Compoundsof the Invention

The present invention relates to compounds of Formula (I) useful asmodulators of CFTR activity:

or pharmaceutically acceptable salts thereof; wherein:

-   -   ring A is selected from:

-   -   R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂;    -   R² is hydrogen, —CH₃, —CF₃, —OH, or —CH₂OH;    -   R³ is hydrogen, —CH₃, —OCH₃, or —CN;    -   provided that both R² and R³ are not simultaneously hydrogen.

COMPOUNDS AND DEFINITIONS

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated.

The term “ABC-transporter” as used herein means an ABC-transporterprotein or a fragment thereof comprising at least one binding domain,wherein said protein or fragment thereof is present in vivo or in vitro.The term “binding domain” as used herein means a domain on theABC-transporter that can bind to a modulator. See, e.g., Hwang, T. C. etal., J. Gen. Physiol. (1998): 111(3), 477-90.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR, R117H CFTR, andG551D CFTR (see, e.g., http://www.genet.sickkids.on.ca/cfte, for CFTRmutations).

The term “modulating” as used herein means increasing or decreasing by ameasurable amount.

The term “normal CFTR” or “normal CFTR function” as used herein meanswild-type like CFTR without any impairment due to environmental factorssuch as smoking, pollution, or anything that produces inflammation inthe lungs.

The term “reduced CFTR” or “reduced CFTR function” as used herein meansless than normal CFTR or less than normal CFTR function.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed. Additionally, generalprinciples of organic chemistry are described in “Organic Chemistry”,Thomas Sorrell, University Science Books, Sausalito: 1999, and “March'sAdvanced Organic Chemistry”, 5^(th) Ed., Ed.: Smith, M. B. and March,J., John Wiley & Sons, New York: 2001, the entire contents of which arehereby incorporated by reference.

Combinations of substituents envisioned by this invention are preferablythose that result in the formation of stable or chemically feasiblecompounds. The term “stable”, as used herein, refers to compounds thatare not substantially altered when subjected to conditions to allow fortheir production, detection, and preferably their recovery,purification, and use for one or more of the purposes disclosed herein.In some embodiments, a stable compound or chemically feasible compoundis one that is not substantially altered when kept at a temperature of40° C. or less, in the absence of moisture or other chemically reactiveconditions, for at least a week.

The term “protecting group”, as used herein, refers to an agent used totemporarily to block one or more desired reactive sites in amultifunctional compound. In certain embodiments, a protecting group hasone or more, or preferably all, of the following characteristics: a)reacts selectively in good yield to give a protected substrate that isstable to the reactions occurring at one or more of the other reactivesites; and b) is selectively removable in good yield by reagents that donot attack the regenerated functional group. Exemplary protecting groupsare detailed in Greene, T. W., Wuts, P. G in “Protective Groups inOrganic Synthesis”, Third Edition, John Wiley & Sons, New York: 1999,and other editions of this book, the entire contents of which are herebyincorporated by reference.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. Unless otherwise stated, alltautomeric forms of the compounds of the invention are within the scopeof the invention; e.g., compounds of Formula (I) may exist as tautomers:

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.Such compounds, particularly compounds that contain deuterium atoms, mayexhibit modified metabolic properties.

Description of Exemplary Compounds:

The present invention provides a compound of Formula (I):

or pharmaceutically acceptable salts thereof, wherein:

-   -   ring A is selected from:

-   -   R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂;    -   R² is hydrogen, —CH₃, —CF₃, —OH, or —CH₂OH;    -   R³ is hydrogen, —CH₃, —OCH₃, or —CN;    -   provided that both R² and R³ are not simultaneously hydrogen.

In one embodiment, ring A is

In one embodiment, ring A is

In another embodiment, ring A is

In yet another embodiment, ring A is

In one embodiment, R¹ is —CF₃.

In another embodiment, R¹ is —CN.

In another embodiment, R¹ is —C≡CCH₂N(CH₃)₂.

In one embodiment, R² is —CH₃.

In another embodiment, R² is —CF₃.

In another embodiment, R² is —OH.

In another embodiment, R² is —CH₂OH.

In one embodiment, R³ is —CH₃.

In one embodiment, R³ is —OCH₃.

In another embodiment, R³ is —CN.

In one embodiment, R² is hydrogen; and R³ is —CH₃, —OCH₃, or —CN.

In another embodiment, R² is —CH₃, —CF₃, —OH, or —CH₂OH; and R³ ishydrogen.

In several embodiments of the present invention, ring A is

R¹ is —CF₃, R² is hydrogen; and R³ is —CH₃, —OCH₃, or —CN. In otherembodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R³ is —CH₃. Or, R³ is —OCH₃. Or, R³is —CN.

In further embodiments of the present invention, ring A is

R¹ is —CF₃, R² is —CH₃, —CF₃, —OH, or —CH₂OH, and R³ is hydrogen. Inother embodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R² is —CH₃. Or, R² is —CF₃. Or, R² is—OH. Or, R² is —CH₂OH.

In several embodiments of the present invention, ring A is

R¹ is —CF₃, R² is hydrogen; and R³ is —CH₃, —OCH₃, or —CN. In otherembodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R³ is —OCH₃. Or, R³ is —CH₃. Or, R³is —CN.

In further embodiments of the present invention, ring A is

R¹ is —CF₃, R² is —CH₃, —CF₃, —OH, or —CH₂OH, and R³ is hydrogen. Inother embodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R² is —CH₃. Or, R² is —CF₃. Or, R² is—OH. Or, R² is —CH₂OH.

In several embodiments of the present invention, ring A is

R¹ is —CF₃, R² is hydrogen; and R³ is —CH₃, —OCH₃, or —CN. In otherembodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R³ is —CH₃. Or, R³ is —OCH₃. Or, R³is —CN.

In further embodiments of the present invention, ring A is

R¹ is —CF₃, R² is —CH₃, —CF₃, —OH, or —CH₂OH, and R³ is hydrogen. Inother embodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R² is —CH₃. Or, R² is —CF₃. Or, R² is—OH. Or, R² is —CH₂OH.

In several embodiments of the present invention, ring A is

R¹ is —CF₃, R² is hydrogen; and R³ is —CH₃, —OCH₃, or —CN. In otherembodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R³ is —CH₃. Or, R³ is —OCH₃. Or, R³is —CN.

In further embodiments of the present invention, ring A is

R¹ is —CF₃, R² is —CH₃, —CF₃, —OH, or —CH₂OH, and R³ is hydrogen. Inother embodiments, R¹ is —CN. In still further embodiments, R¹ is—C≡CCH₂N(CH₃)₂. In one embodiment, R² is —CH₃. Or, R² is —CF₃. Or, R² is—OH. Or, R² is —CH₂OH.

Representative compounds of the present invention are set forth below inTable 1 below.

TABLE 1

1

2

3

4

5

6

7

8

9

10

11

12

13

14 General Synthetic Schemes

Compounds of the present invention are readily prepared by methods knownin the art, and as depicted in Scheme 1-3.

Scheme 1 depicts a convergent approach to the preparation of compoundsof Formula (I) from substituted benzene derivatives 1a and 2a. In theultimate transformation, amide formation via coupling of carboxylic acid1d with amine 2c to give a compound of Formula (I) can be achieved usingeither O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and triethylamine in N,N-dimethyl formamide(DMF) or propyl sulfronic acid cyclic anhydride (T3P®) and pyridine in2-methyltetrahydrofuran. Carboxylic acid 1d is prepared from thecorresponding substituted benzene derivative 1a via a sequencecommencing with heat-mediated condensation of 1a with an appropriatemalonate (CO₂R)₂CH═CH(OR), wherein R is an alkyl group such as methyl,ethyl, or the like, to provide 1b.

Compound 1b is converted to carboxylic acid 1d via a three step sequenceincluding intramolecular cyclization upon heating at reflux in Dowthermor diphenyl ether (step b), followed by removal (if needed) of theblocking halo group (step c) under palladium-catalyzed dehalogenationconditions and acid- or base-catalyzed saponification (step d). Theorder of the deprotection and saponification steps can be reversed;i.e., step c can occur before or after step d, as depicted in Scheme 1.

Referring again to Scheme 1, aniline derivative 2c can be prepared fromnitrobenzene 2a via a three step sequence. Thus, coupling ofnitrobenzene 2a with a cyclic amine

3 as defined herein in the presence of triethylamine provides compound2b. Palladium-catalyzed reduction of 2b provides amine 2c.

Scheme 2 depicts the synthesis of compounds of Formula (I) bearing apropynyl amine sidechain. Thus, coupling of nitrobenzene 2a, wherein Halis bromide, chloride, or the like, with

3 as defined herein in the presence potassium carbonate in DMSO providescompound 4. Palladium-catalyzed coupling of compound 4 withN,N-dimethylprop-2-yn-1-amine, followed by iron or zinc catalyzedreduction of the nitro moiety, provides amine 5. Coupling of amine 5with carboxylic acid 1d provides compound 6 which is a compound ofFormula (I).

Scheme 3 depicts the synthesis of a compound of Formula (I) wherein

3 is 7-azabicyclo[2.2.1]heptane, optionally bearing an exo or endohydroxy group at the 2-position. The hydroxy-substituted adducts(+)-endo-7-azabicyclo[2.2.1]heptan-2-ol,(−)-endo-7-azabicyclo[2.2.1]heptan-2-ol,(+)-exo-7-azabicyclo[2.2.1]heptan-2-ol, and(−)-exo-7-azabicyclo[2.2.1]heptan-2-ol can be prepared using proceduresas described in Fletcher, S. R., et al., “Total Synthesis andDetermination of the Absolute Configuration of Epibatidine,” J. Org.Chem, 59, pp. 1771-1778 (1994). 7-Azabicyclo[2.2.1]heptane itself iscommercially available from Tyger Scientific Inc. 324 Stokes AvenueEwing, N.J., 08638 USA.

Thus, as with the series of transformations summarized in Schemes 1 and2, coupling of compound 2a with the bicyclo[2.2.1]amine 7 providescompound 8. If a hydroxy group is present in compound 8, it may benecessary to protect the hydroxy group with a protecting group prior tosubsequent transformations. Thus, treatment of compound 8 withtert-butyl dimethylsilyl chloride using known conditions provides theprotected compound 9 prior to reduction of the nitro moiety to providethe amine 10. Amide formation with 1d (cf. Scheme 3) and removal of thehydroxy protecting group (as needed) provides compound 11 which is acompound of Formula (I).

Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

In one aspect of the present invention, pharmaceutically acceptablecompositions are provided, wherein these compositions comprise any ofthe compounds as described herein, and optionally comprise apharmaceutically acceptable carrier, adjuvant or vehicle. In certainembodiments, these compositions optionally further comprise one or moreadditional therapeutic agents.

It will also be appreciated that certain of the compounds of presentinvention can exist in free form for treatment, or where appropriate, asa pharmaceutically acceptable derivative or a prodrug thereof. Accordingto the present invention, a pharmaceutically acceptable derivative or aprodrug includes, but is not limited to, pharmaceutically acceptablesalts, esters, salts of such esters, or any other adduct or derivativewhich upon administration to a patient in need thereof is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. A“pharmaceutically acceptable salt” means any non-toxic salt or salt ofan ester of a compound of this invention that, upon administration to arecipient, is capable of providing, either directly or indirectly, acompound of this invention or an inhibitorily active metabolite orresidue thereof.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge, et al. describes pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange.

Other pharmaceutically acceptable salts include adipate, alginate,ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate,butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, edisylate (ethanedisulfonate),ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate,gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersible products may be obtained by such quaternization.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate.

As described above, the pharmaceutically acceptable compositions of thepresent invention additionally comprise a pharmaceutically acceptablecarrier, adjuvant, or vehicle, which, as used herein, includes any andall solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various carriers used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention. Some examples ofmaterials which can serve as pharmaceutically acceptable carriersinclude, but are not limited to, ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, or potassiumsorbate, partial glyceride mixtures of saturated vegetable fatty acids,water, salts or electrolytes, such as protamine sulfate, disodiumhydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zincsalts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, woolfat, sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating or lessening the severity of a condition, disease, or disorderimplicated by CFTR mutation. In certain embodiments, the presentinvention provides a method of treating a condition, disease, ordisorder implicated by a deficiency of the CFTR activity, the methodcomprising administering a composition comprising a compound of Formula(I) to a subject, preferably a mammal, in need thereof.

In certain embodiments, the present invention provides a method oftreating diseases associated with reduced CFTR function due to mutationsin the gene encoding CFTR or environmental factors (e.g., smoke). Thesediseases include, cystic fibrosis, chronic bronchitis, recurrentbronchitis, acute bronchitis, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), female infertility causedby congenital absence of the uterus and vagina (CAUV), idiopathicchronic pancreatitis (ICP), idiopathic recurrent pancreatitis,idiopathic acute pancreatitis, chronic rhinosinusitis, primarysclerosing cholangitis, allergic bronchopulmonary aspergillosis,diabetes, dry eye, constipation, allergic bronchopulmonary aspergillosis(ABPA), bone diseases (e.g., osteoporosis), and asthma.

In certain embodiments, the present invention provides a method fortreating diseases associated with normal CFTR function. These diseasesinclude, chronic obstructive pulmonary disease (COPD), chronicbronchitis, recurrent bronchitis, acute bronchitis, rhinosinusitis,constipation, pancreatitis including chronic pancreatitis, recurrentpancreatitis, and acute pancreatitis, pancreatic insufficiency, maleinfertility caused by congenital bilateral absence of the vas deferens(CBAVD), mild pulmonary disease, idiopathic pancreatitis, liver disease,hereditary emphysema, gallstones, gasgtro-esophageal reflux disease,gastrointestinal malignancies, inflammatory bowel disease, constipation,diabetes, arthritis, osteoporosis, and osteopenia.

In certain embodiments, the present invention provides a method fortreating diseases associated with normal CFTR function includinghereditary hemochromatosis, coagulation-fibrinolysis deficiencies, suchas protein C deficiency, Type 1 hereditary angioedema, lipid processingdeficiencies, such as familial hypercholesterolemia, Type 1chylomicronemia, abetalipoproteinemia, lysosomal storage diseases, suchas I-cell disease/pseudo-Hurler, mucopolysaccharidoses,Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetesinsipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases suchas Alzheimer's disease, Parkinson's disease, amyotrophic lateralsclerosis, progressive supranuclear palsy, Pick's disease, severalpolyglutamine neurological disorders such as Huntington's,spinocerebullar ataxia type I, spinal and bulbar muscular atrophy,dentatorubal pallidoluysian, and myotonic dystrophy, as well asspongiform encephalopathies, such as hereditary Creutzfeldt-Jakobdisease (due to prion protein processing defect), Fabry disease,Straussler-Scheinker syndrome, Gorham's Syndrome, chloridechannelopathies, myotonia congenita (Thomson and Becker forms),Bartter's syndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, PrimaryCiliary Dyskinesia (PCD), PCD with situs inversus (also known asKartagener syndrome), PCD without situs inversus and ciliary aplasia, orSjogren's disease, comprising the step of administering to said mammalan effective amount of a composition comprising a compound of thepresent invention.

According to an alternative preferred embodiment, the present inventionprovides a method of treating cystic fibrosis comprising the step ofadministering to said mammal a composition comprising the step ofadministering to said mammal an effective amount of a compositioncomprising a compound of the present invention.

According to the invention an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective fortreating or lessening the severity of one or more of the diseases,disorders or conditions as recited above.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity of oneor more of the diseases, disorders or conditions as recited above.

In certain embodiments, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who exhibit residual CFTR activity in the apicalmembrane of respiratory and non-respiratory epithelia. The presence ofresidual CFTR activity at the epithelial surface can be readily detectedusing methods known in the art, e.g., standard electrophysiological,biochemical, or histochemical techniques. Such methods identify CFTRactivity using in vivo or ex vivo electrophysiological techniques,measurement of sweat or salivary Cl⁻ concentrations, or ex vivobiochemical or histochemical techniques to monitor cell surface density.Using such methods, residual CFTR activity can be readily detected inpatients heterozygous or homozygous for a variety of differentmutations, including patients homozygous or heterozygous for the mostcommon mutation, ΔF508.

In another embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients who have residual CFTR activity induced oraugmented using pharmacological methods or gene therapy. Such methodsincrease the amount of CFTR present at the cell surface, therebyinducing a hitherto absent CFTR activity in a patient or augmenting theexisting level of residual CFTR activity in a patient.

In one embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients within certain genotypes exhibiting residual CFTRactivity, e.g., class III mutations (impaired regulation or gating),class IV mutations (altered conductance), or class V mutations (reducedsynthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV,and V cystic fibrosis Transmembrane Conductance Regulator Defects andOpportunities of Therapy; Current Opinion in Pulmonary Medicine6:521-529, 2000). Other patient genotypes that exhibit residual CFTRactivity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, the compounds and compositions of the presentinvention are useful for treating or lessening the severity of cysticfibrosis in patients within certain clinical phenotypes, e.g., amoderate to mild clinical phenotype that typically correlates with theamount of residual CFTR activity in the apical membrane of epithelia.Such phenotypes include patients exhibiting pancreatic insufficiency orpatients diagnosed with idiopathic pancreatitis and congenital bilateralabsence of the vas deferens, or mild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or patch), bucally, as an oral or nasal spray,or the like, depending on the severity of the infection being treated.In certain embodiments, the compounds of the invention may beadministered orally or parenterally at dosage levels of about 0.01 mg/kgto about 50 mg/kg and preferably from about 0.5 mg/kg to about 25 mg/kg,of subject body weight per day, one or more times a day, to obtain thedesired therapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms are prepared by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The activity of a compound utilized in this invention as a modulator ofCFTR may be assayed according to methods described generally in the artand in the Examples herein.

It will also be appreciated that the compounds and pharmaceuticallyacceptable compositions of the present invention can be employed incombination therapies, that is, the compounds and pharmaceuticallyacceptable compositions can be administered concurrently with, prior to,or subsequent to, one or more other desired therapeutics or medicalprocedures. The particular combination of therapies (therapeutics orprocedures) to employ in a combination regimen will take into accountcompatibility of the desired therapeutics and/or procedures and thedesired therapeutic effect to be achieved. It will also be appreciatedthat the therapies employed may achieve a desired effect for the samedisorder (for example, an inventive compound may be administeredconcurrently with another agent used to treat the same disorder), orthey may achieve different effects (e.g., control of any adverseeffects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated.”

In one embodiment, the additional agent is selected from a mucolyticagent, a bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent. In a further embodiment, theadditional agent is a CFTR modulator other than a compound of thepresent invention.

In one embodiment, the additional agent is an antibiotic. Exemplaryantibiotics useful herein include tobramycin, including tobramycininhaled powder (TIP), azithromycin, aztreonam, including the aerosolizedform of aztreonam, amikacin, including liposomal formulations thereof,ciprofloxacin, including formulations thereof suitable foradministration by inhalation, levoflaxacin, including aerosolizedformulations thereof, and combinations of two antibiotics, e.g.,fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogenphosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent reduces the activity of theepithelial sodium channel blocker (ENaC) either directly by blocking thechannel or indirectly by modulation of proteases that lead to anincrease in ENaC activity (e.g., seine proteases, channel-activatingproteases). Exemplary such agents include camostat (a trypsin-likeprotease inhibitor), QAU145, 552-02, GS-9411, INO-4995, Aerolytic, andamiloride. Additional agents that reduce the activity of the epithelialsodium channel blocker (ENaC) can be found, for example in PCTPublication No. WO2009/074575, the entire contents of which areincorporated herein in their entirety.

Amongst other diseases described herein, combinations of CFTRmodulators, such as compounds of Formula I, and agents that reduce theactivity of ENaC are use for treating Liddle's syndrome, an inflammatoryor allergic condition including cystic fibrosis, primary ciliarydyskinesia, chronic bronchitis, chronic obstructive pulmonary disease,asthma, respiratory tract infections, lung carcinoma, xerostomia andkeratoconjunctivitis sire, respiratory tract infections (acute andchronic; viral and bacterial) and lung carcinoma.

Combinations of CFTR modulators, such as compounds of Formula I, andagents that reduce the activity of ENaC are also useful for treatingdiseases mediated by blockade of the epithelial sodium channel alsoinclude diseases other than respiratory diseases that are associatedwith abnormal fluid regulation across an epithelium, perhaps involvingabnormal physiology of the protective surface liquids on their surface,e.g., xerostomia (dry mouth) or keratoconjunctivitis sire (dry eye).Furthermore, blockade of the epithelial sodium channel in the kidneycould be used to promote diuresis and thereby induce a hypotensiveeffect.

Asthma includes both intrinsic (non-allergic) asthma and extrinsic(allergic) asthma, mild asthma, moderate asthma, severe asthma,bronchitic asthma, exercise-induced asthma, occupational asthma andasthma induced following bacterial infection. Treatment of asthma isalso to be understood as embracing treatment of subjects, e.g., of lessthan 4 or 5 years of age, exhibiting wheezing symptoms and diagnosed ordiagnosable as “wheezy infants”, an established patient category ofmajor medical concern and now often identified as incipient orearly-phase asthmatics. (For convenience this particular asthmaticcondition is referred to as “wheezy-infant syndrome”.) Prophylacticefficacy in the treatment of asthma will be evidenced by reducedfrequency or severity of symptomatic attack, e.g., of acute asthmatic orbronchoconstrictor attack, improvement in lung function or improvedairways hyperreactivity. It may further be evidenced by reducedrequirement for other, symptomatic therapy, i.e., therapy for orintended to restrict or abort symptomatic attack when it occurs, e.g.,anti-inflammatory (e.g., cortico-steroid) or bronchodilatory.Prophylactic benefit in asthma may, in particular, be apparent insubjects prone to “morning dipping”. “Morning dipping” is a recognizedasthmatic syndrome, common to a substantial percentage of asthmatics andcharacterized by asthma attack, e.g., between the hours of about 4-6 am,i.e., at a time normally substantially distant from any previouslyadministered symptomatic asthma therapy.

Chronic obstructive pulmonary disease includes chronic bronchitis ordyspnea associated therewith, emphysema, as well as exacerbation ofairways hyperreactivity consequent to other drug therapy, in particular,other inhaled drug therapy. In some embodiments, the combinations ofCFTR modulators, such as compounds of Formula I, and agents that reducethe activity of ENaC are useful for the treatment of bronchitis ofwhatever type or genesis including, e.g., acute, arachidic, catarrhal,croupus, chronic or phthinoid bronchitis.

In another embodiment, the additional agent is a CFTR modulator otherthan a compound of formula I, i.e., an agent that has the effect ofmodulating CFTR activity. Exemplary such agents include ataluren(“PTC124®”; 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid),sinapultide, lancovutide, depelestat (a human recombinant neutrophilelastase inhibitor), cobiprostone(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid), or(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid. In another embodiment, the additional agent is(3-(6-(1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamido)-3-methylpyridin-2-yl)benzoicacid.

In another embodiment, the additional agent is a nutritional agent.Exemplary such agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating an implantable medical device, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. Suitable coatingsand the general preparation of coated implantable devices are describedin U.S. Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings aretypically biocompatible polymeric materials such as a hydrogel polymer,polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylacticacid, ethylene vinyl acetate, and mixtures thereof. The coatings mayoptionally be further covered by a suitable topcoat of fluorosilicone,polysaccarides, polyethylene glycol, phospholipids or combinationsthereof to impart controlled release characteristics in the composition.

Another aspect of the invention relates to modulating CFTR activity in abiological sample or a patient (e.g., in vitro or in vivo), which methodcomprises administering to the patient, or contacting said biologicalsample with a compound of Formula (I) or a composition comprising saidcompound. The term “biological sample”, as used herein, includes,without limitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Modulation of CFTR in a biological sample is useful for a variety ofpurposes that are known to one of skill in the art. Examples of suchpurposes include, but are not limited to, the study of CFTR inbiological and pathological phenomena; and the comparative evaluation ofnew modulators of CFTR.

In yet another embodiment, a method of modulating activity of an anionchannel in vitro or in vivo, is provided comprising the step ofcontacting said channel with a compound of Formula (I). In preferredembodiments, the anion channel is a chloride channel or a bicarbonatechannel. In other preferred embodiments, the anion channel is a chloridechannel.

According to an alternative embodiment, the present invention provides amethod of increasing the number of functional CFTR in a membrane of acell, comprising the step of contacting said cell with a compound ofFormula (I).

According to another preferred embodiment, the activity of the CFTR ismeasured by measuring the transmembrane voltage potential. Means formeasuring the voltage potential across a membrane in the biologicalsample may employ any of the known methods in the art, such as opticalmembrane potential assay or other electrophysiological methods.

The optical membrane potential assay utilizes voltage-sensitive FRETsensors described by Gonzalez and Tsien (See, Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells.” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R.Y. Tsien (1997); “Improved indicators of cell membrane potential thatuse fluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission can be monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

In another aspect the present invention provides a kit for use inmeasuring the activity of CFTR or a fragment thereof in a biologicalsample in vitro or in vivo comprising (i) a composition comprising acompound of Formula (I) or any of the above embodiments; and (ii)instructions for a) contacting the composition with the biologicalsample and b) measuring activity of said CFTR or a fragment thereof. Inone embodiment, the kit further comprises instructions for a) contactingan additional composition with the biological sample; b) measuring theactivity of said CFTR or a fragment thereof in the presence of saidadditional compound, and c) comparing the activity of the CFTR in thepresence of the additional compound with the density of the CFTR in thepresence of a composition of Formula (I). In preferred embodiments, thekit is used to measure the density of CFTR.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

Processes and Intermediates for Making Compounds of Formula (I)

Another aspect of the invention relates to a process for preparing acompound of Formula (Ic):

or pharmaceutically acceptable salts thereof, wherein the processcomprises:

(a) reacting the acid of formula 1d with an amine of formula 2c toprovide a compound of Formula (Ic)

wherein:

Ring A is selected from:

wherein

-   -   R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂;    -   R² is hydrogen, —CH₃, —CF₃, —OH, or —CH₂OH;    -   R³ is hydrogen, —CH₃, —OCH₃, or —CN;        -   provided that both R² and R³ are not simultaneously            hydrogen, and    -   R^(a) is hydrogen or a silyl protecting group selected from the        group consisting of trimethylsilyl (TMS),        tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl        (TBDMS), triisopropylsilyl (TIPS), and        [2-(trimethylsilyl)ethoxy]methyl (SEM).

In one embodiment, the reaction of the acid of formula 1d with the amineof formula 2c occurs in a solvent in the presence ofO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and triethylamine or in a solvent in thepresence of propyl phosphonic acid cyclic anhydride (T3P®) and pyridine.More particularly, the solvent comprises N,N-dimethyl formamide, ethylacetate, or 2-methyltetrahydrofuran.

In another embodiment, R^(a) is hydrogen or TBDMS.

In another embodiment, R^(a) is TBDMS.

In another embodiment, the process comprises a further deprotectionstep; for instance, when ring A is

wherein R^(a) is a silyl protecting group, to generate a compound ofFormula (Ic), wherein ring A is

Typically, removal of a silyl protecting group requires treatment withacid such as acetic acid or a dilute mineral acid or the like, althoughother reagents, such as a source of fluoride ion (e.g.,tetrabutylammonium fluoride), may be used.

In the process, the amine of formula 2c is prepared from a compound offormula 2a comprising the steps of:

-   -   (a) reacting the compound of formula 2a with an amine of formula        3 to provide the compound of formula 2b

wherein:

Hal is F, Cl, Br, or I; and

the amine of formula 3 is

and

-   -   (b) reducing the compound of formula 2b to the amine of formula        2c.

In one embodiment of the process for making the amine of formula 2c, theamine of formula 3 in step (a) is generated in situ from thecorresponding quaternary ammonium salt, such as an amine hydrochloridesalt, although other ammonium salts (e.g. the trifluoracetate salt), maybe used as well.

In one embodiment of step (a) for forming the amine of formula 2c, whenthe amine of formula 3 is

R^(a) is hydrogen or TBDMS. More particularly, R^(a) is TBDMS.

In another embodiment, step (a) occurs in a polar aprotic solvent in thepresence of a tertiary amine base. Examples of tertiary amines that canbe employed include triethylamine, diisopropylethyl amine,1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane(DABCO) and pyridine. Examples of solvents that can be employed includeN,N-dimethyl formamide, dimethyl sulfoxide or acetonitrile.

In one embodiment, the tertiary amine base is triethylamine.

In another embodiment, step (a) occurs in acetonitrile in the presenceof triethylamine.

In another embodiment, the reaction temperature of step (a) is betweenapproximately 75° C. and approximately 85° C.

In another embodiment, the reaction time for step (a) is betweenapproximately 2 and approximately 30 hours.

In one embodiment of the process for making the amine of formula 2c,step (b) occurs in a polar protic solvent or a mixture of polar proticsolvents in the presence of a palladium catalyst. When palladium is thecatalyst, the solvent in step (b) typically is a polar protic solventsuch as an alcohol. More particularly, comprises methanol or ethanol.

In another embodiment, step (b) occurs in a polar protic solvent, suchas water, in the presence of Fe and FeSO₄ or Zn and AcOH.

Another aspect of the invention relates to a process for preparing acompound of Formula (Ic):

or pharmaceutically acceptable salts thereof, comprising the steps of

(a) reacting a compound of formula 2a with an amine of formula 3 toprovide a compound of formula 2b

(b) converting the compound of formula 2b to the amine of formula 2c viareduction

and

(c) reacting the amine of formula 2c with an acid of formula 1d toprovide a compound of Formula (Ic)

wherein Hal is F, Cl, Br, or I;

the amine of formula 3 is

and

ring A is selected from:

wherein

-   -   R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂;    -   R² is hydrogen, —CH₃, —CF₃, —OH, or —CH₂OH;    -   R³ is hydrogen, —CH₃, —OCH₃, or —CN;        -   provided that both R² and R³ are not simultaneously            hydrogen, and    -   R^(a) is hydrogen or a silyl protecting group selected from the        group consisting of trimethylsilyl (TMS),        tert-butyldiphenylsilyl (TBDPS), tert-butyldimethylsilyl        (TBDMS), triisopropylsilyl (TIPS), and        [2-(trimethylsilyl)ethoxy]methyl (SEM).

In one embodiment, the amine of formula 3 in step (a) is generated insitu from the corresponding quaternary ammonium salt, such as an aminehydrochloride salt, although other ammonium salts (e.g. thetrifluoracetate salt), may be used as well.

In one embodiment of step (a) for forming the amine of formula 2c, whenthe amine of formula 3 is

R^(a) is hydrogen or TBDMS. More particularly, R^(a) is TBDMS.

In another embodiment, step (a) occurs in a polar aprotic solvent in thepresence of a tertiary amine base. Examples of tertiary amines that canbe employed include triethylamine, diisopropylethyl amine,1,5-diazabicyclo[4.3.0]non-5-ene (DBN),1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclo[2.2.2]octane(DABCO) and pyridine.

In one embodiment, the tertiary amine base is triethylamine.

In another embodiment, step (a) occurs in acetonitrile in the presenceof triethylamine.

In another embodiment, the reaction temperature of step (a) is betweenapproximately 75° C. and approximately 85° C.

In another embodiment, the reaction time for step (a) is betweenapproximately 2 and approximately 30 hours.

In one embodiment of the process for making the amine of formula 2c,step (b) occurs in a polar protic solvent or a mixture of polar proticsolvents in the presence of a palladium catalyst. When palladium is thecatalyst, the solvent in step (b) typically is a polar protic solventsuch as an alcohol. More particularly, the solvent comprises methanol orethanol.

In another embodiment, step (b) occurs in a polar protic solvent, suchas water, in the presence of Fe and FeSO₄ or Zn and AcOH.

In one embodiment of step (c), the reaction of the acid of formula 1dwith the amine of formula 2c occurs in a solvent in the presence ofO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and triethylamine or in a solvent in thepresence of propyl phosphonic acid cyclic anhydride (T3P®) and pyridine.More particularly, the solvent comprises N,N-dimethyl formamide, ethylacetate, or 2-methyltetrahydrofuran.

In another embodiment, R^(a) is hydrogen or TBDMS.

In another embodiment, R^(a) is TBDMS.

In another embodiment, the process comprises a further deprotectionstep; for instance, when ring A is

wherein R^(a) is a silyl protecting group, to generate a compound ofFormula (I), wherein ring A is

Typically, removal of a silyl protecting group requires treatment withacid such as acetic acid or a dilute mineral acid or the like, althoughother reagents, such as a source of fluoride ion (e.g.,tetrabutylammonium fluoride), may be used.

Another aspect of the invention relates to a compound which is

wherein ring A is

wherein

R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂, and

R^(a) is a silyl protecting group selected from the group consisting oftrimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and[2-(trimethylsilyl)ethoxy]methyl (SEM).

Another aspect of the invention relates to a compound which is

wherein ring A is

wherein

R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂, and

R^(a) is a silyl protecting group selected from the group consisting oftrimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and[2-(trimethylsilyl)ethoxy]methyl (SEM).

Another aspect of the invention relates to a compound of Formula (IA):

or pharmaceutically acceptable salts thereof, wherein:

is selected from

wherein

-   -   R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂;    -   R² is hydrogen, —CH₃, —CF₃, —OH, or —CH₂OH;    -   R³ is hydrogen, —CH₃, —OCH₃, or —CN;        -   provided that both R² and R³ are not simultaneously            hydrogen, and    -   R^(a) is a silyl protecting group selected from the group        consisting of trimethylsilyl (TMS), tert-butyldiphenylsilyl        (TBDPS), tert-butyldimethylsilyl (TBDMS), triisopropylsilyl        (TIPS), and [2-(trimethylsilyl)ethoxy]methyl (SEM).

Another aspect of the invention relates to a compound of Formula (I)

or pharmaceutically acceptable salts thereof, wherein:

Ring A is selected from:

wherein

-   -   R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂;    -   R² is hydrogen, —CH₃, —CF₃, —OH, or —CH₂OH;    -   R³ is hydrogen, —CH₃, —OCH₃, or —CN;        -   provided that both R² and R³ are not simultaneously            hydrogen;    -   made by any of the processes disclosed herein.

Another aspect of the invention relates to a compound selected from thegroup consisting of:

made by any of the processes disclosed herein.

EXAMPLES Intermediate 1:4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (17)

The synthesis of the title compound is depicted in Scheme 4.

Preparation of diethyl 2-((2-chloro-5-(trifluoromethyl)phenylamino)methylene) malonate (14)

2-Chloro-5-(trifluoromethyl)aniline 12 (200 g, 1.023 mol), diethyl2-(ethoxymethylene)malonate 13 (276 g, 1.3 mol) and toluene (100 mL)were combined under a nitrogen atmosphere in a three-neck, 1-L roundbottom flask equipped with Dean-Stark condenser. The solution was heatedwith stirring to 140° C. and the temperature was maintained for 4 h. Thereaction mixture was cooled to 70° C. and hexane (600 mL) was slowlyadded. The resulting slurry was stirred and allowed to warm to roomtemperature. The solid was collected by filtration, washed with 10%ethyl acetate in hexane (2×400 mL) and then dried under vacuum toprovide a white solid (350 g, 94% yield) as the desired condensationproduct diethyl 2-((2-chloro-5-(trifluoromethyl)phenylamino) methylene)malonate 14. ¹H NMR (400 MHz, DMSO-d₆) δ 11.28 (d, J=13.0 Hz, 1H), 8.63(d, J=13.0 Hz, 1H), 8.10 (s, 1H), 7.80 (d, J=8.3 Hz, 1H), 7.50 (dd,J=1.5, 8.4 Hz, 1H), 4.24 (q, J=7.1 Hz, 2H), 4.17 (q, J=7.1 Hz, 2H), 1.27(m, 6H).

Preparation of ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate(15)

A 3-neck, 1-L flask was charged with Dowtherm® (200 mL, 8 mL/g), whichwas degassed at 200° C. for 1 h. The solvent was heated to 260° C. andcharged in portions over 10 min with diethyl2-((2-chloro-5-(trifluoromethyl)phenylamino) methylene)malonate 14 (25g, 0.07 mol). The resulting mixture was stirred at 260° C. for 6.5 hours(h) and the resulting ethanol byproduct removed by distillation. Themixture was allowed to slowly cool to 80° C. Hexane (150 mL) was slowlyadded over 30 minutes (min), followed by an additional 200 mL of hexaneadded in one portion. The slurry was stirred until it had reached roomtemperature. The solid was filtered, washed with hexane (3×150 mL), andthen dried under vacuum to provide ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate 15as a tan solid (13.9 g, 65% yield). ¹H NMR (400 MHz, DMSO-d₆) δ 11.91(s, 1H), 8.39 (s, 1H), 8.06 (d, J=8.3 Hz, 1H), 7.81 (d, J=8.4 Hz, 1H),4.24 (q, J=7.1 Hz, 2H), 1.29 (t, J=7.1 Hz, 3H).

Preparation of ethyl4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate (16)

A 3-neck, 5-L flask was charged with of ethyl8-chloro-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylate 15(100 g, 0.3 mol), ethanol (1250 mL, 12.5 mL/g) and triethylamine (220mL, 1.6 mol). The vessel was then charged with 10 g of 10% Pd/C (50%wet) at 5° C. The reaction was stirred vigorously under hydrogenatmosphere for 20 h at 5° C., after which time the reaction mixture wasconcentrated to a volume of approximately 150 mL. The product, ethyl4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate 16, as a slurrywith Pd/C, was taken directly into the next step.

Preparation of4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (17)

Ethyl 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylate 16 (58 g, 0.2mol, crude reaction slurry containing Pd/C) was suspended in NaOH (814mL of 5 M, 4.1 mol) in a 1-L flask with a reflux condenser and heated at80° C. for 18 h, followed by further heating at 100° C. for 5 h. Thereaction was filtered warm through packed Celite to remove Pd/C and theCelite was rinsed with 1 N NaOH. The filtrate was acidified to about pH1 to obtain a thick, white precipitate. The precipitate was filteredthen rinsed with water and cold acetonitrile. The solid was then driedunder vacuum to provide4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid 17 as awhite solid (48 g, 92% yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ 15.26 (s,1H), 13.66 (s, 1H), 8.98 (s, 1H), 8.13 (dd, J=1.6, 7.8 Hz, 1H),8.06-7.99 (m, 2H).

Intermediate 2:4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (21)

The synthesis of the title compound is depicted in Scheme 5.

Preparation of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (20)

To a flask containing 7-azabicyclo[2.2.1]heptane hydrochloride 7a (4.6g, 34.43 mmol, obtained from Tyger Scientific Inc., 324 Stokes Avenue,Ewing, N.J., 08638 USA under a nitrogen atmosphere was added a solutionof 4-fluoro-1-nitro-2-(trifluoromethyl)benzene 18 (6.0 g, 28.69 mmol)and triethylamine (8.7 g, 12.00 mL, 86.07 mmol) in acetonitrile (50 mL).The reaction flask was heated at 80° C. under a nitrogen atmosphere for16 h. The reaction mixture was allowed to cool and then was partitionedbetween water and dichloromethane. The organic layer was washed with 1 MHCl, dried over Na₂SO₄, filtered, and concentrated to dryness.Purification by silica gel chromatography (0-10% ethyl acetate inhexanes) yielded7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane 19 (7.2g, 88% yield) as a yellow solid. ¹H NMR (400.0 MHz, DMSO-d₆) δ 8.03 (d,J=9.1 Hz, 1H), 7.31 (d, J=2.4 Hz, 1H), 7.25 (dd, J=2.6, 9.1 Hz, 1H),4.59 (s, 2H), 1.69-1.67 (m, 4H), 1.50 (d, J=7.0 Hz, 4H).

Preparation of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (20)

A flask charged with7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane 19(7.07 g, 24.70 mmol) and 10% Pd/C (0.71 g, 6.64 mmol) was evacuated andthen flushed with nitrogen. Ethanol (22 mL) was added and the reactionflask was fitted with a hydrogen balloon. After stirring vigorously for12 h, the reaction mixture was purged with nitrogen and Pd/C was removedby filtration. The filtrate was concentrated to a dark oil under reducedpressure and the residue purified by silica gel chromatography (0-15%ethyl acetate in hexanes) to provide4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline 20 as apurple solid (5.76 g, 91% yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ 6.95(dd, J=2.3, 8.8 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.72 (d, J=8.8 Hz, 1H),4.89 (s, 2H), 4.09 (s, 2H), 1.61-1.59 (m, 4H) and 1.35 (d, J=6.8 Hz,4H).

Intermediate 3: 2-amino-5-(7-azabicyclo[2.2.1]heptan-7-yl)benzonitrile(23)

The synthesis of the title compound is depicted in Scheme 6.

Preparation of 5-(7-azabicyclo[2.2.1]heptan-7-yl)-2-nitrobenzonitrile(22)

To a solution of 5-fluoro-2-nitrobenzonitrile 21 (160 mg, 0.96 mmol) inacetonitrile (1 mL) was slowly added 7-azabicyclo[2.2.1]heptanehydrochloride 7a (129 mg, 0.96 mmol) and triethylamine (244 mg, 335.7μL, 2.41 mmol). The reaction was stirred at 60° C. for 4 h. The reactionwas quenched with water, acidified with 1 N HCl to pH 1, and extractedwith dichloromethane (3×10 mL). The combined organic layers were washedwith water, dried over MgSO₄, filtered and concentrated to provide5-(7-azabicyclo[2.2.1]heptan-7-yl)-2-nitrobenzonitrile 22 (205 mg, 87%yield). LC/MS m/z 244.3 [M+H]⁺, retention time 1.69 min (RP-C₁₈, 10-99%CH₃CN/0.05% TFA over 3 min).

Preparation of 2-amino-5-(7-azabicyclo[2.2.1]heptan-7-yl)benzonitrile(23)

A flask charged with5-(7-azabicyclo[2.2.1]heptan-7-yl)-2-nitrobenzonitrile 22 (205 mg,0.8427 mmol) and 10% Pd/C (41 mg, 0.39 mmol) was flushed with nitrogenand then evacuated under vacuum. Methanol (4 mL) was added undernitrogen atmosphere and the flask was fitted with a hydrogen balloon.After stirring for 15 min, the Pd/C was removed by filtration andsolvent was removed under reduced pressure to provide2-amino-5-(7-azabicyclo[2.2.1]heptan-7-yl)benzonitrile 23 (170 mg, 95%yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ 7.02 (dd, J=2.8, 9.0 Hz, 1H), 6.87(d, J=2.7 Hz, 1H), 6.68 (d, J=9.0 Hz, 1H), 5.36 (s, 2H), 4.09 (s, 2H),1.59 (d, J=6.8 Hz, 4H), 1.34 (d, J=6.8 Hz, 4H).

Intermediate 4:4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(3-(dimethylamino)prop-1-ynyl)aniline(27)

The synthesis of the title compound is depicted in Scheme 7.

Preparation of 7-(3-bromo-4-nitrophenyl)-7-azabicyclo[2.2.1]heptane (25)

To a solution of 2-bromo-4-fluoro-1-nitro-benzene 24 (1.1 g, 4.8 mmol)and K₂CO₃ (2.0 g, 14.3 mmol) in DMSO (8.400 mL) was added7-azabicyclo[2.2.1]heptane 7a (765.4 mg, 5.7 mmol) portion-wise. Thereaction was stirred at 80° C. for 24 h. The reaction was diluted withwater to precipitate the product. The solid was redissolved indichloromethane, washed with 1.0 N HCl, dried over MgSO₄, filtered andconcentrated to provide7-(3-bromo-4-nitrophenyl)-7-azabicyclo[2.2.1]heptane 25 (1.1 g, 78%yield). The crude product was used directly in the next step. LC/MS m/z299.1 [M+H]⁺, retention time 1.97 min (RP-C₁₈, 10-99% CH₃CN/0.05% TFAover 3 min).

Preparation of3-[5-(7-azabicyclo[2.2.1]heptan-7-yl)-2-nitro-phenyl]-N,N-dimethyl-prop-2-yn-1-amine(26)

To 7-(3-bromo-4-nitro-phenyl)-7-azabicyclo[2.2.1]heptane 25 (500 mg,1.683 mmol), Pd(PPh₃)₂Cl₂ (59 mg, 0.08 mmol), and cuprous iodide (9.616mg, 1.708 μL, 0.05049 mmol) was added a solution ofN,N-dimethylprop-2-yn-1-amine (420 mg, 538 μL, 5.05 mmol) in degassedDMF (5 mL) and triethylamine (5 mL). The reaction mixture was microwavedunder N₂ for 10 min at 100° C. The reaction was diluted with ethylacetate, washed with 50% saturated sodium bicarbonate solution (2×20mL), water, and brine. The solution was dried over anhydrous Na₂SO₄ andfiltered, leaving a red solid. Purification by silica gel chromatography(0-50% dichloromethane in ethyl acetate) yielded3-[5-(7-azabicyclo[2.2.1]heptan-7-yl)-2-nitro-phenyl]-N,N-dimethyl-prop-2-yn-1-amine26 (400 mg, 79% yield). LC/MS m/z 300.5 [M+H]⁺, retention time 1.11 min(RP-C₁₈, 10-99% CH₃CN/0.05% TFA over 3 min).

Preparation of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(3-dimethylaminoprop-1-ynyl)aniline(27)

3-[5-(7-Azabicyclo[2.2.1]heptan-7-yl)-2-nitro-phenyl]-N,N-dimethyl-prop-2-yn-1-amine26 (340 mg, 1.14 mmol), iron (634 mg, 11.36 mmol) and ferrous sulfateheptahydrate (316 mg, 1.136 mmol) were suspended in water (1 mL) andrefluxed for 20 min. The reaction was filtered and the solid washed withmethanol and dichloromethane. The filtrate was concentrated and purifiedby silica gel chromatography using (0-5% methanol in dichloromethane) toprovide4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(3-dimethylaminoprop-1-ynyl)aniline27 (148 mg, 48% yield). LC/MS m/z 270.3 [M+H]⁺, retention time 0.25 min(RP-C₁₈, 10-99% CH₃CN/0.05% TFA over 3 min).

Intermediate 5:exo-4-(2-(tert-butyldimethylsilyloxy)-7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline(30)

The synthesis of the title compound is depicted in Scheme 8.

Preparation ofexo-7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-ol(28)

To a flask containing exo-7-azabicyclo[2.2.1]heptan-2-ol 7b (0.86 g,5.74 mmol) under a nitrogen atmosphere was added a solution of4-fluoro-1-nitro-2-(trifluoromethyl)benzene 18 (1 g, 4.78 mmol) andtriethylamine (1.45 g, 2.0 mL, 14.35 mmol) in acetonitrile (8 mL). Thereaction was heated at 84° C. under a nitrogen atmosphere for 22 h. Thereaction mixture was allowed to cool and then was partitioned betweenwater and ethyl acetate. The layers were separated and the aqueous layerwas extracted twice with ethyl acetate. The combined organic layers weredried over Na₂SO₄, filtered, and concentrated to dryness. Purificationby silica gel chromatography (0-50% ethyl acetate in hexanes) providedexo-7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-ol28 as a yellow solid (0.67 g, 46% yield). LC/MS m/z 303.3 [M+H]⁺,retention time 1.51 min (RP-C₁₈, 10-99% CH₃CN/0.05% TFA over 3 min).

Preparation ofexo-tert-butyl-dimethyl-[[7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-yl]oxy]silane29

Tert-butyl-chloro-dimethyl-silane (197 mg, 1.267 mmol) was added to asolution of 4H-imidazole (144 mg, 2.11 mmol) in DMF (0.5 mL). After thesolution stopped bubbling,exo-7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-ol28 (255 mg, 0.84 mmol) was added as a solution in DMF (0.6 mL) andstirred at room temperature for 14 h. The reaction was quenched withwater and extracted twice with diethyl ether, dried over MgSO₄, filteredand concentrated to a colorless oil. Purification by silica gelchromatography (0-40% dichloromethane in hexanes) affordedexo-tert-butyl-dimethyl-[[7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-yl]oxy]silane29 (318 mg, 90% yield) as a yellow oil. ¹H NMR (400.0 MHz, DMSO-d₆) δ8.01 (d, J=9.2 Hz, 1H), 7.29 (d, J=2.4 Hz, 1H), 7.19 (dd, J=2.6, 9.2 Hz,1H), 4.60 (t, J=4.4 Hz, 1H), 4.47 (d, J=5.2 Hz, 1H), 4.07 (dd, J=2.0,6.8 Hz, 1H), 1.94 (dd, J=6.4, 12.8 Hz, 1H), 1.71-1.47 (m, 3H), 1.39-1.32(m, 2H), 0.65 (s, 9H), 0.03 (s, 6H).

Preparation ofexo-4-[5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)aniline(30)

A flask containing palladium on activated carbon (10 wt %, 30 mg, 0.28mmol) was evacuated, purged with N₂, and charged with a solution ofexo-tert-butyl-dimethyl-[[7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-yl]oxy]silane29 (301 mg, 0.72 mmol) in ethanol (3 mL). The flask was evacuated andthen was equipped with a balloon of H₂ and stirred for 4 h at roomtemperature. The mixture was filtered and concentrated to dryness toyieldexo-4-[5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)aniline30 (268 mg, 96% yield) as an off-white solid. ¹H NMR (400.0 MHz,DMSO-d₆) δ 6.92 (dd, J=2.4, 8.8 Hz, 1H), 6.77 (d, J=2.6 Hz, 1H), 6.70(d, J=8.8 Hz, 1H), 4.84 (s, 2H), 4.11 (t, J=4.4 Hz, 1H), 3.91-3.89 (m,2H), 1.82 (dd, J=7.1, 12.3 Hz, 1H), 1.54-1.39 (m, 3H), 1.20-1.16 (m,2H), 0.79 (s, 9H), 0.02 (s, 6H).

Intermediate 6:endo-4-(2-(tert-butyldimethylsilyloxy)-7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline(34)

The preparation of the title compound is depicted in Scheme 9.

Preparation of 7-azabicyclo[2.2.1]heptan-5-one (31)

To a solution of oxalyl dichloride (165 mg, 113 μL, 1.27 mmol) indichloromethane (3 mL) under a nitrogen atmosphere at −78° C. was addeda solution of DMSO (199 mg, 180 μL, 2.54 mmol) in dichloromethane (0.7mL) dropwise. The reaction mixture was allowed to stir for 30 min andthen a solution ofexo-7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-ol28 (320 mg, 1.06 mmol) in dichloromethane (2.5 mL) was added dropwise.The reaction was stirred for an additional hour at −78° C., and thentriethylamine (536 mg, 738 μL, 5.30 mmol) was added dropwise and thereaction was warmed to room temperature. The reaction mixture wasdiluted with dichloromethane, partitioned between dichloromethane andwater, and the layers were separated. The aqueous layer was extractedonce more with dichloromethane. The combined organic layers were driedover Na₂SO₄, filtered and concentrated to a yellow oil. Purification bysilica gel chromatography (0-30% ethyl acetate in hexanes) provided7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-one 31(266 mg, 84% yield) as a yellow solid. ¹H NMR (400.0 MHz, DMSO-d₆) δ8.06 (d, J=9.1 Hz, 1H), 7.47 (d, J=2.4 Hz, 1H), 7.39 (dd, J=2.6, 9.1 Hz,1H), 4.98 (t, J=4.5 Hz, 1H), 4.84 (d, J=5.4 Hz, 1H), 2.44 (d, J=3.1 Hz,1H), 2.23 (d, J=16 Hz, 1H), 2.00-1.92 (m, 1H), 1.88-1.70 (m, 2H),1.66-1.60 (m, 1H).

Preparation ofendo-7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-ol(32)

To a solution of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-one 31(261 mg, 0.87 mmol) in THF (11 mL) at −55° C. under a nitrogenatmosphere was added a solution of lithium hydrido-trisec-butyl-boron(1.04 mL of 1 M, 1.04 mmol) dropwise. After 30 min, the reaction mixturewas transferred to an ice water bath and stirring was continued. Thereaction mixture was quenched with methanol (1.2 mL) at 0° C. Thereaction mixture was partitioned between dichloromethane/water,separated and the aqueous layer was extracted twice more withdichloromethane. The organic layers were combined, dried over Na₂SO₄,filtered, and concentrated to dryness. Purification by silica gelchromatography (0-50% ethyl acetate in hexanes) providedendo-7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-ol32 (222 mg, 84% yield) as a yellow solid. ¹H NMR (400.0 MHz, DMSO-d₆) δ8.01 (d, J=9.1 Hz, 1H), 7.27 (d, J=3.0 Hz, 1H), 7.22 (dd, J=2.6, 9.1 Hz,1H), 5.17 (d, J=4.4 Hz, 1H), 4.49 (t, J=4.9 Hz, 1H), 4.44 (t, J=4.5 Hz,1H), 4.16-4.10 (m, 1H), 2.20-2.06 (m, 2H), 1.67-1.44 (m, 3H), 1.09 (dd,J=3.5, 12.4 Hz, 1H).

Preparation ofendo-tert-butyl-dimethyl-[[7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-yl]oxy]silane(33)

Tert-butylchlorodimethylsilane (168 mg, 1.08 mmol) was added to asolution of 4H-imidazole (122 mg, 1.80 mmol) in DMF (425.3 μL). Afterthe solution stopped bubbling,endo-7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-ol32 (217 mg, 0.72 mmol) was added as a solution in DMF (1 mL) and stirredat room temperature for 14 h. The reaction was quenched with water andextracted twice with diethyl ether, dried over MgSO₄, filtered, andconcentrated to a colorless oil. Purification by silica gelchromatography (0-40% dichloromethane in hexanes) affordedendo-tert-butyl-dimethyl-[[7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-yl]oxy]silane33 (251 mg, 84% yield) as a yellow oil. ¹H NMR (400.0 MHz, DMSO-d₆) δ8.01 (d, J=9.1 Hz, 1H), 7.32 (d, J=2.3 Hz, 1H), 7.26 (dd, J=2.5, 9.1 Hz,1H), 4.54-4.51 (m, 2H), 4.29-4.26 (m, 1H), 2.20-2.11 (m, 2H), 1.67-1.45(m, 3H), 1.08 (dd, J=3.2, 12.4 Hz, 1H), 0.88 (s, 9H), 0.07 (d, J=2.6 Hz,6H).

Preparation ofendo-4-[5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)aniline(34)

A flask containing palladium on activated carbon (10 wt %, 24 mg, 0.23mmol) was evacuated and then purged under a nitrogen atmosphere. To thiswas added a solution ofendo-tert-butyl-dimethyl-[[7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptan-5-yl]oxy]silane33 (240 mg, 0.58 mmol) in ethanol (5 mL). The reaction mixture wasevacuated, then equipped with a balloon of H₂ and stirred for 4 h atroom temperature. The mixture was filtered and concentrated to drynessto yieldendo-4-[5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)aniline34 (222 mg, 100% yield) as an off-white solid. ¹H NMR (400.0 MHz,DMSO-d₆) δ 6.95 (dd, J=2.4, 8.8 Hz, 1H), 6.79 (d, J=2.6 Hz, 1H), 6.72(d, J=8.8 Hz, 1H), 4.91 (s, 2H), 4.24-4.19 (m, 1H), 4.06-4.03 (m, 2H),2.12-1.99 (m, 2H), 1.55-1.53 (m, 1H), 1.42-1.36 (m, 2H), 0.96 (dd,J=3.2, 12.2 Hz, 1H), 0.87 (s, 9H), 0.05 (s, 6H).

Example Compound 3N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamide

The preparation of the title compound is depicted in Scheme 10.

To a solution of 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylicacid 17 (9.1 g, 35.39 mmol) and4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline 20 (9.2 g,35.74 mmol) in 2-methyltetrahydrofuran (91.00 mL) was added propylphosphonic acid cyclic anhydride (T3P, 50% solution in ethyl acetate,52.68 mL, 88.48 mmol) and pyridine (5.6 g, 5.73 mL, 70.78 mmol) at roomtemperature. The reaction flask heated at 65° C. for 10 h under anitrogen atmosphere. After cooling to room temperature, the reaction wasthen diluted with ethyl acetate and quenched with saturated Na₂CO₃solution (50 mL). The layers were separated, and the aqueous layer wasextracted twice more with ethyl acetate. The combined organic layerswere washed with water, dried over Na₂SO₄, filtered and concentrated toa tan solid. The crude solid product was slurried in ethylacetate/diethyl ether (2:1), collected by vacuum filtration, and washedtwice more with ethyl acetate/diethyl ether (2:1) to provide the productas a light yellow crystalline powder. The powder was dissolved in warmethyl acetate and absorbed onto Celite. Purification by silica gelchromatography (0-50% ethyl acetate in dichloromethane) providedN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamideas a white crystalline solid (13.5 g, 76% yield). LC/MS m/z 496.0[M+H]⁺, retention time 1.48 min (RP-C₁₈, 10-99% CH₃CN/0.05% TFA over 3min). ¹H NMR (400.0 MHz, DMSO-d₆) δ 13.08 (s, 1H), 12.16 (s, 1H), 8.88(s, 1H), 8.04 (dd, J=2.1, 7.4 Hz, 1H), 7.95-7.88 (m, 3H), 7.22 (dd, 2.5,8.9 Hz, 1H), 7.16 (d, J=2.5 Hz, 1H), 4.33 (s, 2H), 1.67 (d, J=6.9 Hz,4H), 1.44 (d, J=6.9 Hz, 4H).

Example Compound 3 Form A HCl SaltN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidehydrochloride (Form A-HCl)

Preparation of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (19)

4-fluoro-1-nitro-2-(trifluoromethyl)benzene (18) (901 g) was added intoa 30 L jacketed vessel. Sodium carbonate (959.1 g) and 5 Ldimethylsulfoxide (DMSO) was added and the mixture was stirred under anitrogen atmosphere. 7-azabicyclo[2.2.1]heptane hydrochloride (7a)(633.4 g) was added to the vessel in portions. The temperature of themixture was gradually raised to 55° C., and the reaction was monitoredby HPLC. When the substrate was less than 1% AUC, the reaction wasconsidered complete. The mixture was then diluted with 10 vol.2-methyltetrahydrofuran and washed with 5.5 vol. water three times untilno DMSO remained in the aqueous layer as determined by HPLC, to give7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (19) in2-methyltetrahydrofuran (approximately 95% yield).

Preparation of hydrochloride salt of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (20.HCl)

Palladium on carbon (150 g, 5% w/w) was charged into a BüchiHydrogenator (20 L capacity) under a nitrogen atmosphere, followed bythe addition of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (19)(1500 g) and 2-methyltetrahydrofuran (10.5 L, 7 vol). Hydrogen gas wascharged into the closed hydrogenator to a pressure of 0.5 bar. A vacuumwas applied for about 2 min, followed by the introduction of hydrogengas to a pressure of 0.5 bar. This process was repeated 2 times.Hydrogen gas was then continuously charged to the mixture at a pressureof 0.5 bar. The mixture was then stirred at a temperature between 18 and23° C. by cooling the vessel jacket. A vacuum was applied to the vesselwhen no more hydrogen gas was consumed and when there was no furtherexotherm. Nitrogen gas was then charged into the vessel at 0.5 bar and avacuum was reapplied, followed by a second charge of 0.5 bar nitrogengas. When the HPLC of a filtered aliquot showed that none of the7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane (19)remained (e.g., ≦0.5%), the reaction mixture was transferred to areceiving flask under nitrogen atmosphere via a filter funnel using aCelite filter. The Celite filter cake was washed with2-methyltetrahydrofuran (3 L, 2 vol). The washings and filtrate werecharged into a vessel equipped with stirring, temperature control, and anitrogen atmosphere. 4M HCl in 1,4-dioxane (1 vol) was addedcontinuously over 1 h into the vessel at 20° C. The mixture was stirredfor an additional 10 h (or overnight), filtered, and washed with2-methyltetrahydrofuran (2 vol) and dried to generate 1519 g of thehydrochloride salt of4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline (20.HCl)as a white crystalline solid (approximately 97% yield).

Alternative preparation of hydrochloride salt of7-[4-amino-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane(20.HCl)

In a Büchi Hydrogenator (20 L capacity), palladium on carbon (5% w/w)(150 g) was introduced under nitrogen followed by the addition of7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane 19(1500 g) and 2-methyltetrahydrofuran (10.5 L, 7 vol). Hydrogen gas wascharged into the vessel to a pressure of 0.5 bar. A vacuum was brieflyapplied (2 min), followed by the introduction of hydrogen gas to apressure of 0.5 bar. This process was repeated 2 more times, and thenhydrogen gas was charged to the hydrogenator continuously at 0.5 bar,and stirring was commenced. The temperature of the reaction mixture wasmaintained at 18 to 23° C. by cooling the vessel jacket. A vacuum wasapplied to the vessel when no more hydrogen gas was consumed and whenthere was no further exotherm. Nitrogen gas was then charged to thevessel, and a vacuum was re-applied, followed by a nitrogen gas chargeat 0.5 bar. The reaction was deemed complete when an HPLC of a filteredaliquot showed that7-[4-nitro-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptane was notdetected (≦0.5%). The reaction mixture was then filtered through Celite.The remaining slurry was transferred to a receiving flask under nitrogengas via a filter funnel containing a Celite filter. The Celite cake waswashed with 2-methyltetrahydrofuran (3 L, 2 vol). The filtrate and thewashings were transferred to a vessel equipped with a stirringmechanism, temperature control, and a nitrogen atmosphere. 4M HCl in1,4-dioxane (1 vol) was added continuously over 1 h to the vessel at 20°C. The resulting mixture was stirred for an additional 10 h, filteredand washed with 2-methyltetrahydrofuran (2 vol) and dried to generate1519 g of7-[4-amino-3-(trifluoromethyl)phenyl]-7-azabicyclo[2.2.1]heptanehydrochloride (20.HCl) as a white crystalline solid.

N-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidehydrochloride (Form A-HCl)

2-Methyltetrahydrofuran (0.57 L, 1.0 vol) was charged into a 30 Ljacketed reactor vessel, followed by the addition of the hydrochloridesalt of 4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)aniline(20.HCl) (791 g, 2.67 mol) and4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid (17)(573 g, 2.23 mol) and an additional 5.2 L (9.0 vol) of2-methyltetrahydrofuran. Stirring commenced, and T3P in2-methyltetrahydrofuran (2.84 kg, 4.46 mol) was added to the reactionmixture over 15 min. Pyridine (534.0 g, 546.0 mL, 6.68 mol) was thenadded via an addition funnel dropwise over 30 min. The mixture waswarmed to 45° C. over about 30 min and stirred for 12-15 h. HPLCanalysis indicated that4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxylic acid waspresent in an amount less than 2%. The mixture was then cooled to roomtemperature. 2-Methyltetrahydrofuran (4 vol, 2.29 L) was added followedby water (6.9 vol, 4 L), while the temperature was maintained below 30°C. The water layer was removed and the organic layer was carefullywashed twice with NaHCO₃ saturated aqueous solution. The organic layerwas then washed with 10% w/w citric acid (5 vol) and finally with water(7 vol). The mixture was polished filtered and transferred into anotherdry vessel. Seed crystals ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidehydrochloride (Form A-HCl) (3.281 g, 5.570 mmol) from an earlier batchwere added. HCl (g) (10 eq) was bubbled over 2 h and the mixture wasstirred overnight. The resulting suspension was filtered, washed with2-methyltetrahydrofuran (4 vol), suction dried and oven dried at 60° C.until constant weight generating 868 g ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidehydrochloride (Form A-HCl).

Example Compound 3 Form B HCl SaltN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(trifluoromethyl)phenyl)-4-oxo-5-(trifluoromethyl)-1,4-dihydroquinoline-3-carboxamidehydrochloride (Form B-HCl)

2-Methyltetrahydrofuran (100 mL) was charged into a 3-necked flaskhaving a nitrogen atmosphere equipped with a stirrer. Example Compound 3Form A-HCl (Example 3B, 55 g, 0.103 mol) was added to the flask,followed by 349 mL of 2-methyltetrahydrofuran, and stirring commenced.28 mL of water was added into the flask and the flask was warmed to aninternal temperature of 60° C. and stirred for 48 h. The flask wascooled to room temperature and stirred for 1 h. The reaction mixture wasvacuum filtered until the filter cake was dry. The solid filter cake waswashed with 2-methyltetrahydrofuran (4 vol) twice. The solid filter cakeremained under vacuum suction for a period of about 30 minutes and wastransferred to a drying tray. The filter cake was dried to a constantweight under vacuum at 60° C., to give Example Compound 3 Form B-HCl asa white crystalline solid (49 g) (approximately 90% yield).

Example Compound 6 Preparation ofN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-cyanophenyl)-5-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide

The preparation of the title compound is depicted in Scheme 11.

To a solution of 5-methyl-4-oxo-1,4-dihydroquinoline-3-carboxylic acid35 (162 mg, 0.80 mmol) and2-amino-5-(7-azabicyclo[2.2.1]heptan-7-yl)benzonitrile 23 (170 mg, 0.80mmol) in 2-methyltetrahydrofuran (1.5 mL) was added propyl phosphonicacid cyclic anhydride (50% solution in ethyl acetate, 949.5 μL, 1.605mmol) and pyridine (126 mg, 129 μL, 1.60 mmol). The reaction was cappedand heated at 100° C. for 65 min with microwave irradiation. Thereaction was cooled to room temperature, diluted with ethyl acetate (10mL), and quenched with saturated Na₂CO₃ solution (6 mL). The organiclayer was dried over Na₂SO₄, filtered and concentrated. Purification bysilica gel chromatography (0-35% ethyl acetate in dichloromethane)providedN-(4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-cyanophenyl)-5-methyl-4-oxo-1,4-dihydroquinoline-3-carboxamide(157 mg, 49% yield). LC/MS m/z 399.3 [M+H]⁺, retention time 1.47 min(RP-C₁₈, 10-99% CH₃CN/0.05% TFA over 3 min). ¹H NMR (400.0 MHz, DMSO-d₆)δ 12.77 (s, 1H), 12.75 (s, 1H), 8.77 (s, 1H), 8.11 (d, J=9.1 Hz, 1H),7.64-7.60 (m, 1H), 7.55 (d, J=8.0 Hz, 1H), 7.34 (d, J=2.8 Hz, 1H), 7.27(dd, J=2.8, 9.1 Hz, 1H), 7.23 (d, J=7.2 Hz, 1H), 4.32 (s, 2H), 2.91 (s,3H), 1.65 (d, J=7.2 Hz, 4H), 1.42 (d, J=6.8 Hz, 4H).

Example Compound 13N-[4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(3-dimethylaminoprop-1-ynyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide

The preparation of the title compound is depicted in Scheme 12.

To a solution of 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylicacid 17 (19 mg, 0.07 mmol) and4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(3-dimethylaminoprop-1-ynyl)aniline27 (20 mg, 0.07 mmol) in 2-methyltetrahydrofuran (190.9 μL) was addedT3P (118 mg, 0.19 mmol) and pyridine (12 mg, 12 μL, 0.15 mmol). Thereaction was heated at 100° C. for 30 min under microwave irradiation.The reaction was diluted with EtOAc and quenched with saturated aqueousNaHCO₃ (50 mL). The layers were separated, and the aqueous layer wasextracted twice with EtOAc. The combined organics were washed once withwater, dried over Na₂SO₄, filtered and concentrated. The residue waspurified by reverse phase HPLC (0-99% CH₃CN/0.05% TFA) to giveN-[4-(7-azabicyclo[2.2.1]heptan-7-yl)-2-(3-dimethylaminoprop-1-ynyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide(8 mg, 17% yield). LC/MS m/z 509.7 [M+H]⁺, retention time 1.06 min(RP-C₁₈, 10-99% CH₃CN/0.05% TFA over 3 min). ¹H NMR (400.0 MHz, DMSO-d₆)δ 13.23 (d, J=6.8 Hz, 1H), 12.40 (s, 1H), 10.31 (s, 1H), 8.96 (d, J=6.6Hz, 1H), 8.40 (d, J=9.0 Hz, 1H), 8.08-8.06 (m, H), 8.07 (dd, J=1.5 Hz,8.1 Hz, 1H), 8.00-7.95 (m, 2H), 7.15-7.09 (m, 2H), 4.49 (s, 2H), 4.29(s, 2H), 2.94 (s, 6H), 1.67 (d, J=7.2 Hz, 4H), 1.44 (d, J=7.0 Hz, 4H).

Example Compound 5Endo-N-[4-[(5S)-5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide

The preparation of the title compound is depicted in Scheme 13.

Preparation ofendo-N-[4-[(5S)-5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide

To a solution of 4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxylicacid 17 (148 mg, 0.58 mmol),O-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (306 mg, 0.81 mmol) in2-methyltetrahydrofuran (2.2 mL) was addedendo-4-[5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)aniline34 (222 mg, 0.58 mmol) followed by triethylamine (146 mg, 201 μL, 1.44mmol). The reaction mixture was heated at 62° C. for 16 h. The reactionmixture was allowed to cool to room temperature, and partitioned between2-methyltetrahydrofuran/water, separated and the aqueous layer wasextracted once more with 2-methyltetrahydrofuran, the organic layerswere combined, dried over Na₂SO₄, filtered and concentrated to dryness.Purification by silica gel chromatography (0-30% ethyl acetate indichloromethane) affordedendo-N-[4-[(5S)-5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide(285 mg, 79% yield). ¹H NMR (400.0 MHz, DMSO-d₆) δ 13.07 (s, 1H), 12.16(s, 1H), 8.88 (s, 1H), 8.04 (dd, J=2.2, 7.3 Hz, 1H), 7.95-7.89 (m, 3H),7.22 (dd, J=2.4, 8.9 Hz, 1H), 7.16 (d, J=2.6 Hz, 1H), 4.29 (m, 3H),2.16-2.07 (m, 2H), 1.62-1.43 (m, 3H), 1.05-1.01 (m, 1H), 0.89 (s, 9H),0.08 (d, J=1.4 Hz, 6H).

Preparation ofendo-N-[4-[(5S)-5-hydroxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide

Endo-N-[4-[(5S)-5-[tert-butyl(dimethyl)silyl]oxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide(281 mg, 0.45 mmol) was dissolved in 1% HCl/Ethanol (2 mL of 1% w/w)solution and allowed to stir a room temperature for 16 h, resulting in awhite precipitate. The reaction was diluted with diethyl ether andfiltered. The collected solid was dissolved in ethyl acetate/saturatedaqueous NaHCO₃ solution. The layers were separated and the aqueous layerwas extracted once more with ethyl acetate. The organic layers werewashed twice with water, dried over Na₂SO₄, filtered and concentrated todryness to yieldendo-N-[4-[(5S)-5-hydroxy-7-azabicyclo[2.2.1]heptan-7-yl]-2-(trifluoromethyl)phenyl]-4-oxo-5-(trifluoromethyl)-1H-quinoline-3-carboxamide(190 mg, 83%). ¹H NMR (400.0 MHz, DMSO-d₆) δ 13.07 (s, 1H), 12.15 (s,1H), 8.88 (s, 1H), 8.04 (dd, J=2.2, 7.4 Hz, 1H), 7.95-7.88 (m, 3H), 7.19(dd, J=2.4, 9.0 Hz, 1H), 7.12 (d, J=2.6 Hz, 1H), 5.00 (d, J=4.2 Hz, 1H),4.25-4.13 (m, 1H), 4.21-4.19 (m, 1H), 4.16-4.13 (m, 1H), 2.15-2.08 (m,2H), 1.61-1.55 (m, 1H), 1.47-1.44 (m, 2H) and 1.03 (dd, J=3.4, 12.3 Hz,1H).

Analytical data for the compounds of Table 1 is shown below:

TABLE 2 Example LC/MS LC/RT^(a) Compound No. M + 1 minutes NMR 1 456.501.76 ¹H NMR (400.0 MHz, DMSO-d₆) δ 12.94 (d, J = 6.1 Hz, 1H), 12.36 (s,1H), 8.80 (d, J = 6.8 Hz, 1H), 8.13 (s, 1H), 7.98 (d, J = 8.9 Hz, 1H),7.68-7.63 (m, 2H), 7.10 (d, J = 8.5 Hz, 1H), 7.01 (s, 1H), 4.28 (s, 2H),2.48 (s, 3H), 2.02-2.00 (m, 2H), 1.86-1.77 (m, 5H), 1.45-1.42 (m, 1H),1.31 (d, J = 11.1 Hz, 2H). 2 458.20 1.20 ¹H NMR (400.0 MHz, DMSO-d₆) δ12.89 (s, 1H), 12.43 (s, 1H), 8.79 (s, 1H), 8.00 (d, J = 8.9 Hz, 1H),7.72-7.68 (m, 2H), 7.44 (dd, J = 2.9, 9.0 Hz, 1H), 7.22 (dd, J = 2.5,9.0 Hz, 1H), 7.15 (d, J = 2.6 Hz, 1H), 4.33 (s, 2H), 3.91 (s, 3H), 1.67(d, J = 6.9 Hz, 4H), 1.43 (d, J = 6.9 Hz, 4H). 3 496.0 1.48 ¹H NMR(400.0 MHz, DMSO-d₆) δ 13.08 (s, 1H), 12.16 (s, 1H), 8.88 (s, 1H), 8.04(dd, J = 2.1, 7.4 Hz, 1H), 7.95-7.88 (m, 3H), 7.22 (dd, 2.5, 8.9 Hz,1H), 7.16 (d, J = 2.5 Hz, 1H), 4.33 (s, 2H), 1.67 (d, J = 6.9 Hz, 4H),1.44 (d, J = 6.9 Hz, 4H). 4 458.50 1.22 ¹H NMR (400.0 MHz, DMSO-d₆) δ13.12 (d, J = 6.7 Hz, 1H), 12.50 (s, 1H), 8.78 (d, J = 6.8 Hz, 1H), 8.10(d, J = 9.1 Hz, 1H), 7.78-7.72 (m, 3H), 7.65 (d, J = 7.7 Hz, 1H), 7.39(d, J = 7.3 Hz, 1H), 7.33 (s, 1H), 5.20 (s, 2H), 4.47 (s, 2H), 1.74 (d,J = 6.7 Hz, 4H), 1.51 (d, J = 7.1 Hz, 4H). 5 512.50 1.55 ¹H NMR (400.0MHz, DMSO-d₆) δ 13.07 (s, 1H), 12.15 (s, 1H), 8.88 (s, 1H), 8.04 (dd, J= 2.2, 7.4 Hz, 1H), 7.95-7.88 (m, 3H), 7.19 (dd, J = 2.4, 9.0 Hz, 1H),7.12 (d, J = 2.6 Hz, 1H), 5.00 (d, J = 4.2 Hz, 1H), 4.25-4.13 (m, 1H),4.21-4.19 (m, 1H), 4.16-4.13 (m, 1H), 2.15-2.08 (m, 2H), 1.61-1.55 (m,1H), 1.47-1.44 (m, 2H) and 1.03 (dd, J = 3.4, 12.3 Hz, 1H). 6 399.301.47 ¹H NMR (400.0 MHz, DMSO-d₆) δ 12.77 (s, 1H), 12.75 (s, 1H), 8.77(s, 1H), 8.11 (d, J = 9.1 Hz, 1H), 7.64-7.60 (m, 1H), 7.55 (d, J = 8.0Hz, 1H), 7.34 (d, J = 2.8 Hz, 1H), 7.27 (dd, J = 2.8, 9.1 Hz, 1H), 7.23(d, J = 7.2 Hz, 1H), 4.32 (s, 2H), 2.91 (s, 3H), 1.65 (d, J = 7.2 Hz,4H), 1.42 (d, J = 6.8 Hz, 4H). 7 453.0 1.62 ¹H NMR (400.0 MHz, DMSO-d₆)δ 13.28 (d, J = 6.4 Hz, 1H), 12.07 (s, 1H), 8.95 (d, J = 6.5 Hz, 1H),8.69 (s, 1H), 8.16 (dd, J = 1.5, 8.7 Hz, 1H), 8.01 (d, J = 8.8 Hz, 1H),7.91 (d, J = 8.7 Hz, 1H), 7.28 (d, J = 7.8 Hz, 1H), 7.22 (s, 1H), 4.38(s, 2H), 1.69 (d, J = 6.4 Hz, 4H), 1.46 (d, J = 6.9 Hz, 4H). 8 512.101.35 ¹H NMR (400.0 MHz, DMSO-d₆) δ 13.07 (s, 1H), 12.13 (s, 1H), 8.88(s, 1H), 8.05-8.02 (m, 1H), 7.95-7.86 (m, 3H), 7.18 (d, J = 9.0 Hz, 1H),7.12 (d, J = 2.5 Hz, 1H), 4.74 (d, J = 5.2 Hz, 1H), 4.33 (m, 1H), 4.11(m, 1H), 3.77 (m, 1H), 1.82 (dd, J = 7.3, 12.5 Hz, 1H), 1.56-1.48 (m,3H), 1.25 (m, 2H). 9 442.10 1.20 ¹H NMR (400.0 MHz, DMSO-d₆) δ 12.84 (s,1H), 12.43 (s, 1H), 8.81 (s, 1H), 8.12 (s, 1H), 8.00 (d, J = 8.9 Hz,1H), 7.64 (m, 2H), 7.21 (dd, J = 2.5, 9.0 Hz, 1H), 7.15 (d, J = 2.6 Hz,1H), 4.32 (s, 2H), 2.47 (s, 3H), 1.67 (d, J = 7.4 Hz, 4H), 1.43 (d, J =6.9 Hz, 4H). 10 442.10 1.40 ¹H NMR (400.0 MHz, DMSO-d₆) δ 12.68 (s, 1H),12.41 (s, 1H), 8.75 (s, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.63-7.60 (m,1H), 7.54 (d, J = 7.8 Hz, 1H), 7.23-7.20 (m, 2H), 7.15 (d, J = 2.7 Hz,1H), 4.33 (s, 2H), 2.89 (s, 3H), 1.67 (d, J = 6.7 Hz, 4H), 1.44 (d, J =6.9 Hz, 4H). 11 444.0 1.30 ¹H NMR (400.0 MHz, DMSO-d₆) δ 13.55 (s, 1H),13.31 (d, J = 7.2 Hz, 1H), 11.58 (s, 1H), 8.86 (d, J = 6.9 Hz, 1H), 8.01(d, J = 9.0 Hz, 1H), 7.66 (t, J = 8.2 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H),7.23 (s, 1H), 7.17 (d, J = 7.7 Hz, 1H), 6.80 (d, J = 7.5 Hz, 1H), 4.39(s, 2H), 1.69 (d, J = 7.3 Hz, 4H), 1.46 (d, J = 7.0 Hz, 4H). 12 510.51.95 ¹H NMR (400.0 MHz, DMSO-d₆) δ 13.16 (d, J = 5.7 Hz, 1H), 12.07 (s,1H), 8.87 (d, J = 6.6 Hz, 1H), 8.05 (dd, J = 2.1, 7.3 Hz, 1H), 7.96-7.92(m, 2H), 7.87 (d, J = 9.0 Hz, 1H), 7.09 (d, J = 9.1 Hz, 1H), 7.00 (s,1H), 4.28 (s, 2H), 2.02-2.00 (m, 2H), 1.88-1.73 (m, 5H), 1.45-1.42 (m,1H), 1.31 (d, J = 11.6 Hz, 2H). 13 509.7 1.06 ¹H NMR (400.0 MHz,DMSO-d₆) δ 13.23 (d, J = 6.8 Hz, 1H), 12.40 (s, 1H), 10.31 (s, 1H), 8.96(d, J = 6.6 Hz, 1H), 8.40 (d, J = 9.0 Hz, 1H), 8.08-8.06 (m, H), 8.07(dd, J = 1.5 Hz, 8.1 Hz, 1H), 8.00-7.95 (m, 2H), 7.15-7.09 (m, 2H), 4.49(s, 2H), 4.29 (s, 2H), 2.94 (s, 6H), 1.67 (d, J = 7.2 Hz, 4H), 1.44 (d,J = 7.0 Hz, 4H). 14 455.7 1.04 ¹H NMR (400.0 MHz, DMSO-d₆) δ 12.63 (s,2H), 8.75 (s, 1H), 8.38 (d, J = 8.8 Hz, 1H), 7.62-7.58 (m, 1H), 7.52 (d,J = 8.1 Hz, 1H), 7.21 (d, J = 7.2 Hz, 1H), 6.96-6.93 (m, 2H), 4.24 (s,2H), 3.70 (s, 2H), 2.92 (s, 3H), 2.28 (s, 6H), 1.65 (d, J = 7.0 Hz, 4H),1.39 (d, J = 6.8 Hz, 4H). ^(a)Retention Time

Assays for Detecting and Measuring ΔF508-CFTR Potentiation Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The assay utilizes fluorescent voltage sensing dyes to measure changesin membrane potential using a fluorescent plate reader (e.g., FLIPR III,Molecular Devices, Inc.) as a readout for increase in functionalΔF508-CFTR in NIH 3T3 cells. The driving force for the response is thecreation of a chloride ion gradient in conjunction with channelactivation by a single liquid addition step after the cells havepreviously been treated with compounds and subsequently loaded with avoltage sensing dye.

Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. This HTS assay utilizes fluorescent voltagesensing dyes to measure changes in membrane potential on the FLIPR IIIas a measurement for increase in gating (conductance) of ΔF508 CFTR intemperature-corrected ΔF508 CFTR NIH 3T3 cells. The driving force forthe response is a Cl⁻ ion gradient in conjunction with channelactivation with forskolin in a single liquid addition step using afluorescent plate reader such as FLIPR III after the cells havepreviously been treated with potentiator compounds (or DMSO vehiclecontrol) and subsequently loaded with a redistribution dye.

Solutions

Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1, HEPES 10,pH 7.4 with NaOH.

Chloride-Free Bath Solution: Chloride Salts in Bath Solution #1 areSubstituted with Gluconate Salts.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at ˜20,000/well in 384-wellmatrigel-coated plates and cultured for 2 hrs at 37° C. before culturingat 27° C. for 24 hrs. for the potentiator assay. For the correctionassays, the cells are cultured at 27° C. or 37° C. with and withoutcompounds for 16-24 hours. Electrophysiological Assays for assayingΔF508-CFTR modulation properties of compounds.

Using Chamber Assay

Using chamber experiments were performed on polarized airway epithelialcells expressing ΔF508-CFTR to further characterize the ΔF508-CFTRmodulators identified in the optical assays. Non-CF and CF airwayepithelia were isolated from bronchial tissue, cultured as previouslydescribed (Galietta, L. J. V., Lantero, S., Gazzolo, A., Sacco, O.,Romano, L., Rossi, G. A., & Zegarra-Moran, O. (1998) In Vitro Cell. Dev.Biol. 34, 478-481), and plated onto Costar® Snapwell™ filters that wereprecoated with NIH3T3-conditioned media. After four days the apicalmedia was removed and the cells were grown at an air liquid interfacefor >14 days prior to use. This resulted in a monolayer of fullydifferentiated columnar cells that were ciliated, features that arecharacteristic of airway epithelia. Non-CF HBE were isolated fromnon-smokers that did not have any known lung disease. CF-HBE wereisolated from patients homozygous for ΔF508-CFTR.

HBE grown on Costar® Snapwell™ cell culture inserts were mounted in anUsing chamber (Physiologic Instruments, Inc., San Diego, Calif.), andthe transepithelial resistance and short-circuit current in the presenceof a basolateral to apical gradient (I_(SC)) were measured using avoltage-clamp system (Department of Bioengineering, University of Iowa,Iowa). Briefly, HBE were examined under voltage-clamp recordingconditions (V_(hold)=0 mV) at 37° C. The basolateral solution contained(in mM) 145 NaCl, 0.83 K₂HPO₄, 3.3 KH₂PO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10Glucose, 10 HEPES (pH adjusted to 7.35 with NaOH) and the apicalsolution contained (in mM) 145 NaGluconate, 1.2 MgCl₂, 1.2 CaCl₂, 10glucose, 10 HEPES (pH adjusted to 7.35 with NaOH).

Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. Forskolin (10μM) and all test compounds were added to the apical side of the cellculture inserts. The efficacy of the putative ΔF508-CFTR potentiatorswas compared to that of the known potentiator, genistein.

Patch-Clamp Recordings

Total CF current in ΔF508-NIH3T3 cells was monitored using theperforated-patch recording configuration as previously described (Rae,J., Cooper, K., Gates, P., & Watsky, M. (1991) J. Neurosci. Methods 37,15-26). Voltage-clamp recordings were performed at 22° C. using anAxopatch 200B patch-clamp amplifier (Axon Instruments Inc., Foster City,Calif.). The pipette solution contained (in mM) 150 N-methyl-D-glucamine(NMDG)-Cl, 2 MgCl₂, 2 CaCl₂, 10 EGTA, 10 HEPES, and 240 μg/mLamphotericin-B (pH adjusted to 7.35 with HCl). The extracellular mediumcontained (in mM) 150 NMDG-Cl, 2 MgCl₂, 2 CaCl₂, 10 HEPES (pH adjustedto 7.35 with HCl). Pulse generation, data acquisition, and analysis wereperformed using a PC equipped with a Digidata 1320 A/D interface inconjunction with Clampex 8 (Axon Instruments Inc.). To activateΔF508-CFTR, 10 μM forskolin and 20 μM genistein were added to the bathand the current-voltage relation was monitored every 30 sec.

Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in IΔ_(F508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

Single-Channel Recordings

Gating activity of wt-CFTR and temperature-corrected ΔF508-CFTRexpressed in NIH3T3 cells was observed using excised inside-out membranepatch recordings as previously described (Dalemans, W., Barbry, P.,Champigny, G., Jallat, S., Dott, K., Dreyer, D., Crystal, R. G.,Pavirani, A., Lecocq, J-P., Lazdunski, M. (1991) Nature 354, 526-528)using an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.).The pipette contained (in mM): 150 NMDG, 150 aspartic acid, 5 CaCl₂, 2MgCl₂, and 10 HEPES (pH adjusted to 7.35 with Tris base). The bathcontained (in mM): 150 NMDG-Cl, 2 MgCl₂, 5 EGTA, 10 TES, and 14 Trisbase (pH adjusted to 7.35 with HCl). After excision, both wt- andΔF508-CFTR were activated by adding 1 mM Mg-ATP, 75 nM of the catalyticsubunit of cAMP-dependent protein kinase (PKA; Promega Corp. Madison,Wis.), and 10 mM NaF to inhibit protein phosphatases, which preventedcurrent rundown. The pipette potential was maintained at 80 mV. Channelactivity was analyzed from membrane patches containing ≦2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Compounds of the invention are useful as modulators of ATP bindingcassette transporters. Examples of activities and efficacies of thecompounds of Formula (I) are shown below in Table 3. The compoundactivity is illustrated with “+++” if activity was measured to be lessthan 2.0 μM, “++” if activity was measured to be from 2 μM to 5.0 μM,“+” if activity was measured to be greater than 5.0 μM, and “−” if nodata was available. The efficacy is illustrated with “+++” if efficacywas calculated to be greater than 100%, “++” if efficacy was calculatedto be from 100% to 25%, “+” if efficacy was calculated to be less than25%, and “−” if no data was available. It should be noted that 100%efficacy is the maximum response obtained with4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)phenol.

TABLE 3 Example Activity Compound No. EC₅₀ (μm) % Efficacy 1 +++ ++ 2+++ ++ 3 +++ ++ 4 +++ ++ 5 +++ +++ 6 +++ +++ 7 +++ ++ 8 +++ ++ 9 +++ ++10 +++ +++ 11 +++ ++ 12 +++ ++ 13 +++ ++ 14 +++ ++

What is claimed is:
 1. A process for preparing a compound of Formula(Ic):

or pharmaceutically acceptable salts thereof, wherein the processcomprises: (a) reacting the acid of formula 1d with an amine of formula2c to provide a compound of Formula (Ic)

wnerein: ring A is selected from:

wherein R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂; R² is hydrogen, —CH₃, —CF₃,—OH, or —CH₂OH; R³ is hydrogen, —CH₃, —OCH₃, or —CN; provided that bothR² and R³ are not simultaneously hydrogen, and R^(a) is hydrogen or asilyl protecting group selected from the group consisting oftrimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),tert-butyldimethylsilyl (TBDMS) triisopropylsilyl (TIPS) and[2-(trimethylsilyl)ethoxy]methyl (SEM).
 2. The process of claim 1,wherein the reaction of the acid of formula 1d with the amine of formula2c occurs in a solvent in the presence ofO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and triethylamine or in a solvent in thepresence of propyl phosphonic acid cyclic anhydride (T3P®) and pyridine.3. The process of claim 2, wherein the solvent comprises N,N-dimethylformamide, ethyl acetate, or 2-methyltetrahydrofuran.
 4. The process ofclaim 1, wherein R^(a) is hydrogen or TBDMS.
 5. The process of claim 1,wherein R^(a) is TBDMS.
 6. The process of claim 1 further comprising adeprotection step to remove the silyl protecting group when ring A is

wherein R^(a) is a silyl protecting group, to generate a compound ofFormula (I), wherein ring A is


7. The process of claim 1, wherein the amine of formula 2c is preparedfrom a compound of formula 2a comprising the steps of: (a) reacting thecompound of formula 2a with an amine of formula 3 to provide thecompound of formula 2b

wherein: Hal is F, Cl, Br, or I; and the amine of formula 3 is

 and (b) reducing the compound of formula 2b to the amine of formula 2c


8. The process of claim 7, wherein the amine of formula 3 in step (a) isgenerated in situ from the amine hydrochloride salt.
 9. The process ofclaim 7, wherein R^(a) is hydrogen or TBDMS.
 10. The process of claim 7,wherein R^(a) is TBDMS.
 11. The process of claim 7, wherein step (a)occurs in a polar aprotic solvent in the presence of a tertiary aminebase.
 12. The process of claim 11, wherein step (a) occurs inacetonitrile in the presence of triethylamine.
 13. The process of claim7, wherein the reaction temperature of step (a) is between approximately75° C. and approximately 85° C.
 14. The process of claim 7, wherein thereaction time is between approximately 2 and approximately 30 hours. 15.The process of claim 7, wherein step (b) occurs in a polar proticsolvent in the presence of a palladium catalyst.
 16. The process ofclaim 15, wherein the solvent in step (b) comprises methanol or ethanol.17. The process of claim 7, wherein step (b) occurs in a polar proticsolvent in the presence of Fe and FeSO₄ or Zn and AcOH.
 18. The processof claim 17, wherein the polar protic solvent is water.
 19. A processfor preparing a compound of Formula (Ic),

or pharmaceutically acceptable salts thereof, comprising the steps of(a) reacting a compound of formula 2a with an amine of formula 3 toprovide a compound of formula 2b

(b) converting the compound of formula 2b to the amine of formula 2c viareduction

 and (c) reacting the amine of formula 2c with an acid of formula 1d toprovide a compound of Formula (Ic)

wherein Hal is F, Cl, Br, or I; the amine of formula 3 is

 and ring A is selected from:

wherein R¹ is —CF₃, —CN, or —C≡CCH₂N(CH₃)₂; R² is hydrogen, —CH₃, —CF₃,—OH, or —CH₂OH; R³ is hydrogen, —CH₃, —OCH₃, or —CN; provided that bothR² and R³ are not simultaneously hydrogen, and R^(a) is hydrogen or asilyl protecting group selected from the group consisting oftrimethylsilyl (TMS), tert-butyldiphenylsilyl (TBDPS),tert-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS), and[2-(trimethylsilyl)ethoxy]methyl (SEM).
 20. The process of claim 19,wherein the amine of formula 3 in step (a) is generated in situ from theamine hydrochloride salt.
 21. The process of claim 20, wherein R^(a) ishydrogen or TBDMS.
 22. The process of claim 21, wherein R^(a) is TBDMS.23. The process of claim 19, wherein step (a) occurs in a polar aproticsolvent in the presence of a tertiary amine base.
 24. The process ofclaim 23, wherein step (a) occurs in acetonitrile in the presence oftriethylamine.
 25. The process of claim 19, wherein the reactiontemperature of step (a) is between approximately 75° C. andapproximately 85° C.
 26. The process of claim 19, wherein the reactiontime is between approximately 2 and approximately 30 hours.
 27. Theprocess of claim 19, wherein step (b) occurs in a polar protic solventin the presence of a palladium catalyst.
 28. The process of claim 27,wherein the solvent in step (b) comprises methanol or ethanol.
 29. Theprocess of claim 19, wherein step (b) occurs in a polar protic solventin the presence of Fe and FeSO₄ or Zn and AcOH.
 30. The process of claim19, wherein the polar protic solvent is water.
 31. The process of claim19, wherein step (c) occurs in a solvent in the presence ofO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) and triethylamine or in a solvent in thepresence of propyl phosphonic acid cyclic anhydride (T3P®) and pyridine.32. The process of claim 31, wherein the solvent in step (c) comprisesN,N-dimethyl formamide (DMF), ethyl acetate, or 2-methyltetrahydrofuran.33. The process of claim 31, wherein R^(a) is hydrogen or TBDMS.
 34. Theprocess of claim 33, wherein R^(a) is TBDMS.
 35. The process of claim 19further comprising a deprotection reaction when ring A is

 wherein R^(a) is a silyl protecting group, to generate a compound ofFormula (I), wherein ring A is